Methods and compositions for inhibition of membrane fusion-associated events, including HIV transmission

ABSTRACT

The present invention relates to peptides which exhibit potent anti-retroviral activity. The peptides of the invention comprise DP178 (SEQ ID:1) peptide corresponding to amino acids 638 to 673 of the HIV-1&lt;SUB&gt;LAI &lt;/SUB&gt;gp41 protein, and fragments, analogs and homologs of DP178. The invention further relates to the uses of such peptides as inhibitory of human and non-human retroviral, especially HIV, transmission to uninfected cells.

This is a Continuation-In-Part of Ser. No. 08/360,107 filed Dec. 20,1994, which is a Continuation-In-Part of Ser. No. 08/255,208 filed Jun.7, 1994, which is a Continuation-In-Part of Ser. No. 08/073,028 filedJun. 7, 1993, each of which is incorporated herein by reference in itsentirety.

This invention was made with Government support under Grant No.AI-30411-02 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

1. INTRODUCTION

The present invention relates, first, to DP178 (SEQ ID NO:1), a peptidecorresponding to amino acids 638 to 673 of the HIV-1_(LAI) transmembraneprotein (TM) gp41, and portions or analogs of DP178 (SEQ ID NO:1), whichexhibit anti-membrane fusion capability, antiviral activity, such as theability to inhibit HIV transmission to uninfected CD-4⁺ cells, or anability to modulate intracellular processes involving coiled-coilpeptide structures. Further, the invention relates to the use of DP178(SEQ ID NO:1) and DP178 portions and/or analogs as antifusogenic orantiviral compounds or as inhibitors of intracellular events involvingcoiled-coil peptide structures. The present invention also relates topeptides analogous to DP107 (SEQ ID NO:25), a peptide corresponding toamino acids 558 to 595 of the HIV-1_(LAI) transmembrane protein (TM)gp41, having amino acid sequences present in other viruses, such asenveloped viruses, and/or other organisms, and further relates to theuses of such peptides. These peptides exhibit anti-membrane fusioncapability, antiviral activity, or the ability to modulate intracellularprocesses involving coiled-coil peptide structures. The presentinvention additionally relates to methods for identifying compounds thatdisrupt the interaction between DP178 and DP107, and/or betweenDP107-like and DP178-like peptides. Further, the invention relates tothe use of the peptides of the invention as diagnostic agents. Forexample, a DP178 peptide may be used as an HIV subtype-specificdiagnostic. The invention is demonstrated, first, by way of an Examplewherein DP178 (SEQ ID:1), and a peptide whose sequence is homologous toDP178 are each shown to be potent, non-cytotoxic inhibitors of HIV-1transfer to uninfected CD-4⁺ cells. The invention is furtherdemonstrated by Examples wherein peptides having structural and/or aminoacid motif similarity to DP107 and DP178 are identified in a variety ofviral and nonviral organisms, and in examples wherein a number of suchidentified peptides derived from several different viral systems aredemonstrated to exhibit antiviral activity.

2. BACKGROUND OF THE INVENTION 2.1 Membrane Fusion Events

Membrane fusion is a ubiquitous cell biological process (for a review,see White, J. M., 1992, Science 258:917-924). Fusion events whichmediate cellular housekeeping functions, such as endocytosis,constitutive secretion, and recycling of membrane components, occurcontinuously in all eukaryotic cells.

Additional fusion events occur in specialized cells. Intracellularly,for example, fusion events are involved in such processes as occur inregulated exocytosis of hormones, enzymes and neurotransmitters.Intercellularly, such fusion events feature prominently in, for example,sperm-egg fusion and myoblast fusion.

Fusion events are also associated with disease states. For example,fusion events are involved in the formation of giant cells duringinflammatory reactions, the entry of all enveloped viruses into cells,and, in the case of human immunodeficiency virus (HIV), for example, areresponsible for the virally induced cell-cell fusion which leads to celldeath.

2.2. The Human Immunodeficiency Virus

The human immunodeficiency virus (HIV) has been implicated as theprimary cause of the slowly degenerative immune system disease termedacquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F. et al.,1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503).There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F.et al., 1983, Science 220:868-870; Gallo R. et al., 1984, Science224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 233:343-346;Guyader, M. et al., 1987, Nature 326:662-669). Further, a large amountof genetic heterogeneity exists within populations of each of thesetypes. Infection of human CD-4⁺ T-lymphocytes with an HIV virus leads todepletion of the cell type and eventually to opportunistic infections,neurological dysfunctions, neoplastic growth, and ultimately death.

HIV is a member of the lentivirus family of retroviruses (Teich, N. etal., 1984, RNA Tumor Viruses, Weiss, R. et al., eds., CSH-Press, pp.949-956). Retroviruses are small enveloped viruses that contain adiploid, single-stranded RNA genome, and replicate via a DNAintermediate produced by a virally-encoded reverse transcriptase, anRNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439).Other retroviruses include, for example, oncogenic viruses such as humanT-cell leukemia viruses (HTLV-I, -II, -III), and feline leukemia virus.

The HIV viral particle consists of a viral core, composed of capsidproteins, that contains the viral RNA genome and those enzymes requiredfor early replicative events. Myristylated Gag protein forms an outerviral shell around the viral core, which is, in turn, surrounded by alipid membrane enveloped derived from the infected cell membrane. TheHIV enveloped surface glycoproteins are synthesized as a single 160 Kdprecursor protein which is cleaved by a cellular protease during viralbudding into two glycoproteins, gp41 and gp120. gp41 is a transmembraneprotein and gp120 is an extracellular protein which remainsnon-covalently associated with gp41, possibly in a trimeric ormultimeric form (Hammarskjold, M. and Rekosh, D., 1989, Biochem.Biophys. Acta 989:269-280).

HIV is targeted to CD-4⁺ cells because the CD-4 cell surface proteinacts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al.,1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768;Maddon et al., 1986, Cell 47:333-348). Viral entry into cells isdependent upon gp120 binding the cellular CD-4+receptor molecules(McDougal, J. S. et al., 1986, Science 231:382-385; Maddon, P. J. etal., 1986, Cell 47:333-348) and thus explains HIV's tropism for CD-4⁺cells, while gp41 anchors the enveloped glycoprotein complex in theviral membrane.

2.3. HIV Treatment

HIV infection is pandemic and HIV associated diseases represent a majorworld health problem. Although considerable effort is being put into thesuccessful design of effective therapeutics, currently no curativeanti-retroviral drugs against AIDS exist. In attempts to develop suchdrugs, several stages of the HIV life cycle have been considered astargets for therapeutic intervention (Mitsuya, H. et al., 1991, FASEB J.5:2369-2381). For example, virally encoded reverse transcriptase hasbeen one focus of drug development. A number ofreverse-transcriptase-targeted drugs, including 2′,3′-dideoxynucleosideanalogs such as AZT, ddI, ddC, and d4T have been developed which havebeen shown to been active against HIV (Mitsuya, H. et al., 1991, Science249:1533-1544). While beneficial, these nucleoside analogs are notcurative, probably due to the rapid appearance of drug resistant HIVmutants (Lander, B. et al., 1989, Science 243:1731-1734). In addition,the drugs often exhibit toxic side effects such as bone marrowsuppression, vomiting, and liver function abnormalities.

Attempts are also being made to develop drugs which can inhibit viralentry into the cell, the earliest stage of HIV infection. Here, thefocus has thus far been on CD4, the cell surface receptor for HIV.Recombinant soluble CD4, for example, has been shown to inhibitinfection of CD-4+T-cells by some HIV-1 strains (Smith, D. H. et al.,1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however,are relatively less sensitive to inhibition by recombinant CD-4 (Daar,E. et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition,recombinant soluble CD-4 clinical trials have produced inconclusiveresults (Schooley, R. et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J.O. et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R. et al., 1989,Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).

The late stages of HIV replication, which involve crucial virus-specificsecondary processing of certain viral proteins, have also been suggestedas possible anti-HIV drug targets. Late stage processing is dependent onthe activity of a viral protease, and drugs are being developed whichinhibit this protease (Erickson, J., 1990, Science 249:527-533). Theclinical outcome of these candidate drugs is still in question.

Attention is also being given to the development of vaccines for thetreatment of HIV infection. The HIV-1 enveloped proteins (gp160, gp120,gp41) have been shown to be the major antigens for anti-HIV antibodiespresent in AIDS patients (Barin, et al., 1985, Science 228:1094-1096).Thus far, therefore, these proteins seem to be the most promisingcandidates to act as antigens for anti-HIV vaccine development. To thisend, several groups have begun to use various portions of gp160gp120,and/or gp41 as immunogenic targets for the host immune system. See forexample, Ivanoff, L. et al., U.S. Pat. No. 5,141,867; Saith, G. et al.,WO 92/22,654; Shafferman, A., WO 91/09,872; Formoso, C. et al., WO90/07,119. Clinical results concerning these candidate vaccines,however, still remain far in the future.

Thus, although a great deal of effort is being directed to the designand testing of anti-retroviral drugs, a truly effective, non-toxictreatment is still needed.

3. SUMMARY OF THE INVENTION

The present invention relates, first, to DP178 (SEQ ID:1), a 36-aminoacid synthetic peptide corresponding to amino acids 638 to 673 of thetransmembrane protein (TM) gp41 from the HIV-1 isolate LAI(HIV-1_(LAI)), which exhibits potent anti-HIV-1 activity. As evidencedby the Example presented below, in Section 6, the DP178 (SEQ ID:1)antiviral activity is so high that, on a weight basis, no other knownanti-HIV agent is effective at concentrations as low as those at whichDP178 (SEQ ID:1) exhibits its inhibitory effects.

The invention further relates to those portions and analogs of DP178which also show such antiviral activity, and/or show anti-membranefusion capability, or an ability to modulate intracellular processesinvolving coiled-coil peptide structures. The term “DP178 analog” refersto a peptide which contains an amino acid sequence corresponding to theDP178 peptide sequence present within the gp41 protein of HIV-1_(LAI),but found in viruses and/or organisms other than HIV-1_(LAI). Such DP178analog peptides may, therefore, correspond to DP178-like amino acidsequences present in other viruses, such as, for example, envelopedviruses, such as retroviruses other than HIV-1_(LAI), as well asnon-enveloped viruses. Further, such analogous DP178 peptides may alsocorrespond to DP178-like amino acid sequences present in nonviralorganisms.

The invention further relates to peptides DP107 (SEQ ID NO:25) analogs.DP107 is a peptide corresponding to amino acids 558-595 of theHIV-1_(LAI) transmembrane protein (TM) gp41. The term “DP107 analog” asused herein refers to a peptide which contains an amino acid sequencecorresponding to the DP107 peptide sequence present within the gp41protein of HIV-1_(LAI), but found in viruses and organisms other thanHIV-1_(LAI). Such DP107 analog peptides may, therefore, correspond toDP107-like amino acid sequences present in other viruses, such as, forfor example, enveloped viruses, such as retroviruses other thanHIV-1_(LAI), as well as non-enveloped viruses. Further, such DP107analog peptides may also correspond to DP107-like amino acid sequencespresent in nonviral organisms.

Further, the peptides of the invention include DP107 analog and DP178analog peptides having amino acid sequences recognized or identified bythe 107×178×4, ALLMOTI5 and/or PLZIP search motifs described herein.

The peptides of the invention may, for example, exhibit antifusogenicactivity, antiviral activity, and/or may have the ability to modulateintracellular processes which involve coiled-coil peptide structures.With respect to the antiviral activity of the peptides of the invention,such an antiviral activity includes, but is not limited to theinhibition of HIV transmission to uninfected CD-4+ cells. Additionally,the antifusogenic capability, antiviral activity or intracellularmodulatory activity of the peptides of the invention merely requires thepresence of the peptides of the invention, and, specifically, does notrequire the stimulation of a host immune response directed against suchpeptides.

The peptides of the invention may be used, for example, as inhibitors ofmembrane fusion-asociated events, such as, for example, the inhibitionof human and non-human retroviral, especially HIV, transmission touninfected cells. It is further contemplated that the peptides of theinvention may be used as modulators of intracellular events involvingcoiled-coil peptide structures.

The peptides of the invention may, alternatively, be used to identifycompounds which may themselves exhibit antifusogenic, antiviral, orintracellular modulatory activity. Additional uses include, for example,the use of the peptides of the invention as organism or viral typeand/or subtype-specific diagnostic tools.

The terms “antifusogenic” and “anti-membrane fusion”, as used herein,refer to an agent's ability to inhibit or reduce the level of membranefusion events between two or more moieties relative to the level ofmembrane fusion which occurs between said moieties in the absence of thepeptide. The moieties may be, for example, cell membranes or viralstructures, such as viral envelopes or pili. The term “antiviral”, asused herein, refers to the compound's ability to inhibit viral infectionof cells, via, for example, cell-cell fusion or free virus infection.Such infection may involve membrane fusion, as occurs in the case ofenveloped viruses, or some other fusion event involving a viralstructure and a cellular structure (e.g., such as the fusion of a viralpilus and bacterial membrane during bacterial conjugation).

It is also contemplated that the peptides of the invention may exhibitthe ability to modulate intracellular events involving coiled-coilpeptide structures. “Modulate”, as used herein, refers to a stimulatoryor inhibitory effect on the intracellular process of interest relativeto the level or activity of such a process in the absence of a peptideof the invention.

Embodiments of the invention are demonstrated below wherein an extremelylow concentration of DP178 (SEQ ID:1), and very low concentrations of aDP178 homolog (SEQ ID:3) are shown to be potent inhibitors of HIV-1mediated CD-4⁺ cell-cell fusion (i.e., syncytial formation) andinfection of CD-4⁺ cells by cell-free virus. Further, it is shown thatDP178 (SEQ ID:1) is not toxic to cells, even at concentrations 3 logshigher than the inhibitory DP-178 (SEQ ID:1) concentration.

The present invention is based, in part, on the surprising discoverythat the DP107 and DP178 domains of the HIV gp41 protein non-covalentlycomplex with each other, and that their interaction is required for thenormal infectivity of the virus. This discovery is described in theExample presented, below, in Section 8. The invention, therefore,further relates to methods for identifying antifusogenic, includingantiviral, compounds that disrupt the interaction between DP107 andDP178, and/or between DP107-like and DP178-like peptides.

Additional embodiments of the invention (specifically, the Examplespresents in Sections 9-16 and 19-25, below) are demonstrated, below,wherein peptides, from a variety of viral and nonviral sources, havingstructural and/or amino acid motif similarity to DP107 and DP178 areidentified, and search motifs for their identification are described.Further, Examples (in Sections 17, 18, 25-29) are presented wherein anumber of the peptides of the invention are demonstrated exhibitsubstantial antiviral activity or activity predictive of antiviralactivity.

3.1. Definitions

Peptides are defined herein as organic compounds comprising two or moreamino acids covalently joined by peptide bonds. Peptides may be referredto with respect to the number of constituent amino acids, i.e., adipeptide contains two amino acid residues, a tripeptide contains three,etc. Peptides containing ten or fewer amino acids may be referred to asoligopeptides, while those with more than ten amino acid residues arepolypeptides. Such peptides may also include any of the modificationsand additional amino and carboxy groups as are described herein.

Peptide sequences defined herein are represented by one-letter symbolsfor amino acid residues as follows:

A (alanine)

R (arginine)

N (asparagine)

D (aspartic acid)

C (cysteine)

Q (glutamine)

E (glutamic acid)

G (glycine)

H (histidine)

I (isoleucine)

L (leucine)

K (lysine)

M (methionine)

F (phenylalanine)

P (proline)

S (serine)

T (threonine)

W (tryptophan)

Y (tyrosine)

V (valine)

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino acid sequence of DP178 (SEQ ID:1) derived from HIV_(LAI);DP178 homologs derived from HIV-1_(SP2) (DP-185; SEQ ID:3), HIV-1_(RF)(SEQ ID:4), and HIV-1_(MN) (SEQ ID:5); DP178 homologs derived from aminoacid sequences of two prototypic HIV-2 isolates, namely, HIV-2_(rod)(SEQ ID:6) and HIV-2_(NTHZ) (SEQ ID:7); control peptides: DP-180 (SEQID:2), a peptide incorporating the amino acid residues of DP178 in ascrambled sequence; DP-118 (SEQ ID:10) unrelated to DP178, whichinhibits HIV-1 cell free virus infection; DP-125 (SEQ ID:8), unrelatedto DP178, also inhibits HIV-1 cell free virus infection; DP-116 (SEQID:9), unrelated to DP178, is negative for inhibition of HIV-1 infectionwhen tested using a cell-free virus infection assay. Throughout thefigures, the one letter amino acid code is used.

FIG. 2. Inhibition of HIV-1 cell-free virus infection by syntheticpeptides. IC₅₀ refers to the concentration of peptide that inhibits RTproduction from infected cells by 50% compared to the untreated control.Control: the level of RT produced by untreated cell cultures infectedwith the same level of virus as treated cultures.

FIG. 3. Inhibition of HIV-1 and HIV-2 cell-free virus infection by thesynthetic peptide DP178 (SEQ ID:1). IC₅₀: concentration of peptide thatinhibits RT production by 50% compared to the untreated control.Control: Level of RT produced by untreated cell cultures infected withthe same level of virus as treated cultures.

FIG. 4A-4B. Fusion Inhibition Assays. FIG. 4A: DP178 (SEQ ID:1)inhibition of HIV-1 prototypic isolate-mediated syncytial formation;data represents the number of virus-induced syncytial per cell. FIG. 4B:DP-180 (SEQ ID:2) represents a scrambled control peptide; DP-185 (SEQID:3) represents a DP178 homolog derived from HIV-1_(SF2) isolate;Control, refers to the number of syncytial produced in the absence ofpeptide.

FIG. 5. Fusion inhibition assay: HIV-1 vs. HIV-2. Data represents thenumber of virus-induced syncytial per well. ND: not done.

FIG. 6. Cytotoxicity study of DP178 (SEQ ID:1) and DP-116 (SEQ ID:9) onCEM cells. Cell proliferation data is shown.

FIG. 7. Schematic representation of HIV-gp41 and maltose binding protein(MBP)-gp41 fusion proteins. DP107 and DP178 are synthetic peptides basedon the two putative helices of gp41. The letter P in the DP107 boxesdenotes an Ile to Pro mutation at amino acid number 578. Amino acidresidues are numbered according to Meyers et al., “Human Retrovirusesand AIDS”, 1991, Theoret. Biol. and Biophys. Group, Los Alamos Natl.Lab., Los Alamos, N. Mex. The proteins are more fully described, below,in Section 8.1.1.

FIG. 8. A point mutation alters the conformation and anti-HIV activityof M41.

FIG. 9. Abrogation of DP178 anti-HIV activity. Cell fusion assays werecarried out in the presence of 10 nM DP178 and various concentrations ofM41Δ178 or M41PΔ178.

FIG. 10. Binding of DP178 to leucine zipper of gp41 analyzed by FAb-DELISA.

FIG. 11A-B. Models for a structural transition in the HIV-1 TM protein.Two models are proposed which indicate a structural transition from anative oligomer to a fusogenic state following a trigger event (possiblygp120 binding to CD4). Common features of both models include (1) thenative state is held together by noncovalent protein-proteininteractions to form the heterodimer of gp120/41 and other interactions,principally though gp41 interactive sites, to form homo-oligomers on thevirus surface of the gp120/41 complexes; (2) shielding of thehydrophobic fusogenic peptide at the N-terminus (F) in the native state;and (3) the leucine zipper domain (DP107) exists as a homo-oligomercoiled coil only in the fusogenic state. The major differences in thetwo models include the structural state (native or fusogenic) in whichthe DP107 and DP178 domains are complexed to each other. In the firstmodel (FIG. 11A) this interaction occurs in the native state and in thesecond (FIG. 11B), it occurs during the fusogenic state. When triggered,the fusion complex in the model depicted in (A) is generated throughformation of coiled-coil interactions in homologous DP107 domainsresulting in an extended α-helix. This conformational change positionsthe fusion peptide for interaction with the cell membrane. In the secondmodel (FIG. 11B), the fusogenic complex is stabilized by the associationof the DP178 domain with the DP107 coiled-coil.

FIG. 12. Motif design using heptad repeat positioning of amino acids ofknown coiled-coils.

FIG. 13. Motif design using proposed heptad repeat positioning of aminoacids of DP107 and DP178.

FIG. 14. Hybrid motif design crossing GCN4 and DP107.

FIG. 15. Hybrid motif design crossing GCN4 and DP178.

FIG. 16. Hybrid motif design 107×178×4, crossing DP107 and DP178. Thismotif was found to be the most consistent at identifying relevantDP107-like and DP178-like peptide regions.

FIG. 17. Hybrid motif design crossing GCN4, DP107, and DP178.

FIG. 18. Hybrid motif design ALLMOTI5 crossing GCN4, DP107, DP178, c-Fosc-Jun, c-Myc, and Flu Loop 36.

FIG. 19. PLZIP motifs designed to identify N-terminal proline-leucinezipper motifs.

FIG. 20. Search results for HIV-1 (BRU isolate) enveloped protein gp41.Sequence search motif designations: Spades (

): 107×178×4; Hearts (♥) ALLMOTI5; Clubs (

): PLZIP; Diamonds (♦): transmembrane region (the putative transmembranedomains were identified using a PC/Gene program designed to search forsuch peptide regions). Asterisk (*): Lupas method. The amino acidsequences identified by each motif are bracketed by the respectivecharacters. Representative sequences chosen based on 107×178×4 searchesare underlined and in bold. DP107 and DP178′ sequences are marked, andadditionally double-underlined and italicized.

FIG. 21. Search results for human respiratory syncytial virus (RSV)strain A2 fusion glycoprotein F1. Sequence search motif designations areas in FIG. 20.

FIG. 22. Search results for simian immunodeficiency virus (SIV)enveloped protein gp41 (AGM3 isolate). Sequence search motifdesignations are as in FIG. 20.

FIG. 23. Search results for canine distemper virus (strainOnderstepoort) fusion glycoprotein 1. Sequence search motif designationsare as in FIG. 20.

FIG. 24. Search results for newcastle disease virus (strainAustralia-Victoria/32) fusion glycoprotein F1. Sequence search motifdesignations are as in FIG. 20.

FIG. 25. Search results for human parainfluenza 3 virus (strain NIH47885) fusion glycoprotein F1. Sequence search motif designations are asin FIG. 20.

FIG. 26. Search results for influenza A virus (strain A/AICHI/2/68)hemagglutinin precursor HA2. Sequence search designations are as in FIG.20.

FIG. 27A-D. Respiratory Syncytial Virus (RSV) peptide antiviral andcircular dichroism data. FIG. 27A-B: Peptides derived from the F2DP178/DP107-like region. Antiviral and CD data. FIG. 27C-D: Peptidesderived from the F1 DP107-like region. Peptide and CD data.

Antiviral activity (AV) is represented by the following qualitativesymbols:

“−”, negative antiviral activity;

“+/−”, antiviral activity at greater than 100 μg/ml;

“+”, antiviral activity at between 50-100 μg/ml;

“++” antiviral activity at between 20-50 μg/ml;

“+++”, antiviral activity at between 1-20 μg/ml;

“++++”, antiviral activity at <1 μg/ml.

CD data, referring to the level of helicity is represented by thefollowing qualitative symbol:

“−”, no helicity;

“+”, 25-50% helicity;

“++”, 50-75% helicity;

“+++”, 75-100% helicity.

IC₅₀ refers to the concentration of peptide necessary to produce only50% of the number of syncytial relative to infected control culturescontaining no peptide. IC₅₀ values were obtained using purified peptidesonly.

FIG. 28A-B. Respiratory Syncytial Virus (RSV) DP178-like region (F1)peptide antiviral and CD data. Antiviral symbols, CD symbols, and IC₅₀are as in FIG. 27A-D. IC₅₀ values were obtained using purified peptidesonly.

FIG. 29A-B. Peptides derived from the HPIV3 F1 DP107-like region.Peptide antiviral and CD data. Antiviral symbols, CD symbols, and IC₅₀are as in FIG. 27A-D. Purified peptides were used to obtain IC₅₀ values,except where the values are marked by an asterisk (*), in which cases,the IC₅₀ values were obtained using a crude peptide preparation.

FIG. 29C. HPIV3 peptide T-184 CD spectrum at 1° C. in 0.1M NaCl 10 mMKPO₄, pH 7.0. The data demonstrates the peptide's helical secondarystructure (θ_(222/208)=1.2) over a wide range of concentrations(100-1500 μM). This evidence is consistent with the peptide forming ahelical coiled-coil structure.

FIG. 30A-B. Peptides derived from the HPIV3 F1 DP178-like region.Peptide antiviral and CD data.

Antiviral symbols, CD symbols, and IC₅₀ are as in FIG. 27A-D. Purifiedpeptides were used to obtain IC₅₀ values, except where the values aremarked by an asterisk (*), in which cases, the IC₅₀ values were obtainedusing a crude peptide preparation.

FIG. 31. Motif search results for simian immunodeficiency virus (SIV)isolate MM251, enveloped polyprotein gp41. Sequence search designationsare as in FIG. 20.

FIG. 32. Motif search results for Epstein-Barr Virus (Strain B95-8),glycoprotein gp110 precursor (designated gp115). BALF4. Sequence searchdesignations are as in FIG. 20.

FIG. 33. Motif search results for Epstein-Barr Virus (Strain B95-8),BZLF1 trans-activator protein (designated EB1 or Zebra). Sequence searchdesignations are as in FIG. 20. Additionally, “@” refers to a well knownDNA binding domain and “+” refers to a well known dimerization domain,as defined by Flemington and Speck (Flemington, E. and Speck, S. H.,1990, Proc. Natl. Acad. Sci. USA 87:9459-9463).

FIG. 34. Motif search results for measles virus (strain Edmonston),fusion glycoprotein F1. Sequence search designations are as in FIG. 20.

FIG. 35. Motif search results for Hepatitis B Virus (Subtype AYW), majorsurface antigen precursor S. Sequence search designations are as in FIG.20.

FIG. 36. Motif search results for simian Mason-Pfizer monkey virus,enveloped (TM) protein gp20. Sequence search designations are as in FIG.20.

FIG. 37. Motif search results for Pseudomonas aerginosa, fimbrialprotein (Pilin). Sequence search designations are as in FIG. 20.

FIG. 38. Motif search results for Neisseria gonorrhoeae fimbrial protein(Pilin). Sequence search designations are as in FIG. 20.

FIG. 39. Motif search results for Hemophilus influenzae fimbrialprotein. Sequence search designations are as in FIG. 20.

FIG. 40. Motif search results for Staphylococcus aureus, toxic shocksyndrome toxin-1. Sequence search designations are as in FIG. 20.

FIG. 41. Motif search results for Staphylococcus aureus enterotoxin TypeE. Sequence search designations are as in FIG. 20.

FIG. 42. Motif search results for Staphylococcus aureus enterotoxin A.Sequence search designations are as in FIG. 20.

FIG. 43. Motif search results for Escherichia coli, heat labileenterotoxin A. Sequence search designations are as in FIG. 20.

FIG. 44. Motif search results for human c-fos proto-oncoprotein.Sequence search designations are as in FIG. 20.

FIG. 45. Motif search results for human lupus KU autoantigen proteinP70. Sequence search designations are as in FIG. 20.

FIG. 46. Motif search results for human zinc finger protein 10. Sequencesearch designations are as in FIG. 20.

FIG. 47. Measles virus (MeV) fusion protein DP178-like region antiviraland CD data. Antiviral symbols, CD symbols, and IC₅₀ are as in FIG.27A-D. IC₅₀ values were obtained using purified peptides.

FIG. 48. Simian immunodeficiency virus (SIV) TM (fusion) proteinDP178-like region antiviral data. Antiviral symbols are as in FIG. 27A-D“NT”, not tested.

FIG. 49A-C. DP178-derived peptide antiviral data. The peptides listedherein were derived from the region surrounding the HIV-1 BRU isolateDP178 region (e.g., gp41 amino acid residues 615-717).

In instances where peptides contained DP178 point mutations, the mutatedamino acid residues are shown with a shaded background. In instances inwhich the test peptide has had an amino and/or carboxy-terminal groupadded or removed (apart from the standard amido- and acetyl-blockinggroups found on such peptides), such modifications are indicated. FIG.49A: The column to the immediate right of the name of the test peptideindicates the size of the test peptide and points out whether thepeptide is derived from a one amino acid peptide “walk” across the DP178region. The next column to the right indicates whether the test peptidecontains a point mutation, while the column to its right indicateswhether certain amino acid residues have been added to or removed fromthe DP178-derived amino acid sequence. FIG. 49B: The column to theimmediate right of the test peptide name indicates whether the peptiderepresents a DP178 truncation, the next column to the right points outwhether the peptide contains a point mutation, and the column to itsright indicates whether the peptide contains amino acids which have beenadded to or removed from the DP178 sequence itself. FIG. 49C: The columnto the immediate right of the test peptide name indicates whether thetest peptide contains a point mutation, while the column to its rightindicates whether amino acid residues have been added to or removed fromthe DP178 sequence itself. IC₅₀ is as defined in FIG. 27A-D, and IC₅₀values were obtained using purified peptides except where marked with anasterisk (*), in which case the IC₅₀ was obtained using a crude peptidepreparation.

FIG. 50. DP107 and DP107 gp41 region truncated peptide antiviral data.IC₅₀ as defined in FIG. 27A-D, and IC₅₀ values were obtained usingpurified peptides except where marked with an asterisk (*), in whichcase the IC₅₀ was obtained using a crude peptide preparation.

FIG. 51A-B. Epstein-Barr virus Strain B95-8 BZLF1 DP178/DP107 analogregion peptide walks and electrophoretic mobility shift assay results.The peptides (T-423 to T-446, FIG. 51A; T-447 to T-461, FIG. 51B)represent one amino acid residue “walks” through the EBV Zebra proteinregion from amino acid residue 173 to 246.

The amino acid residue within this region which corresponds to the firstamino acid residue of each peptide is listed to the left of eachpeptide, while the amino acid residue within this region whichcorresponds to the last amino acid residue of each peptide is listed tothe right of each peptide. The length of each test peptide is listed atthe far right of each line, under the heading “Res”.

“ACT” refers to a test peptide's ability to inhibit Zebra binding to itsresponse element. “+” refers to a visible, but incomplete, abrogation ofthe response element/Zebra homodimer complex; “+++” refers to a completeabrogation of the complex; and “−” represents a lack of complexdisruption.

FIG. 52A-B. Hepatitis B virus subtype AYW major surface antigenprecursor S protein DP178/DP107 analog region and peptide walks. 52Adepicts Domain I (S protein amino acid residues 174-220), which containsa potential DP178/DP107 analog region. In addition, peptides are listedwhich represent one amino acid peptide “walks” through domain I. 52Bdepicts Domain II (S protein amino acid residues 233-291), whichcontains a second potential DP178/DP107 analog region. In addition,peptides are listed which represent one amino acid peptide “walks”through domain II.

5. DETAILED DESCRIPTION OF THE INVENTION

Described herein are peptides which may exhibit antifusogenic activity,antiviral capability, and/or the ability to modulate intracellularprocesses involving coiled-coil peptide structures. The peptidesdescribed include, first, DP178 (SEQ ID NO:1), a gp41-derived 36 aminoacid peptide and fragments and analogs of DP178.

In addition, the peptides of the invention described herein includepeptides which are DP107 analogs. DP107 (SEQ ID NO:25) is a 38 aminoacid peptide corresponding to residues 558 to 595 of the HIV-1_(LAI)transmembrane (TM) gp41 protein. Such DP107 analogs may exhibitantifusogenic capability, antiviral activity or an ability to modulateintracellular processes involving coiled-coil structures.

Further, peptides of the invention include DP107 and DP178 are describedherein having amino acid sequences recognized by the 107×178×4,ALLMOTI5, and PLZIP search motifs. Such motifs are also discussed.

Also described here are antifusogenic, antiviral, intracellularmodulatory, and diagnostic uses of the peptides of the invention.Further, procedures are described for the use of the peptides of theinvention for the identification of compounds exhibiting antifusogenic,antiviral or intracellular modulatory activity.

While not limited to any theory of operation, the following model isproposed to explain the potent anti-HIV activity of DP178, based, inpart, on the experiments described in the Examples, infra. In the HIVprotein, gp41, DP178 corresponds to a putative α-helix region located inthe C-terminal end of the gp41 ectodomain, and appears to associate witha distal site on gp41 whose interactive structure is influenced by theleucine zipper motif, a coiled-coil structure, referred to as DP107. Theassociation of these two domains may reflect a molecular linkage or“molecular clasp” intimately involved in the fusion process. It is ofinterest that mutations in the C-terminal α-helix motif of gp41 (i.e.,the D178 domain) tend to enhance the fusion ability of gp41, whereasmutations in the leucine zipper region (i.e., the DP107 domain) decreaseor abolish the fusion ability of the viral protein. It may be that theleucine zipper motif is involved in membrane fusion while the C-terminalα-helix motif serves as a molecular safety to regulate the availabilityof the leucine zipper during virus-induced membrane fusion.

On the basis of the foregoing, two models are proposed of gp41-mediatedmembrane fusion which are schematically shown in FIG. 11A-B. The reasonfor proposing two models is that the temporal nature of the interactionbetween the regions defined by DP107 and DP178 cannot, as yet, bepinpointed. Each model envisions two conformations for gp41-one in a“native” state as it might be found on a resting virion. The other in a“fusogenic” state to reflect conformational changes triggered followingbinding of gp120 to CD4 and just prior to fusion with the target cellmembrane. The strong binding affinity between gp120 and CD4 may actuallyrepresent the trigger for the fusion process obviating the need for a pHchange such as occurs for viruses that fuse within intracellularvesicles. The two major features of both models are: (1) the leucinezipper sequences (DP107) in each chain of oligomeric enveloped are heldapart in the native state and are only allowed access to one another inthe fusogenic state so as to form the extremely stable coiled-coils, and(2) association of the DP178 and DP107 sites as they exist in gp41 occureither in the native or fusogenic state. FIG. 11A depicts DP178/DP107interaction in the native state as a molecular clasp. On the other hand,if one assumes that the most stable form of the enveloped occurs in thefusogenic state, the model in FIG. 11B can be considered.

When synthesized as peptides, both DP107 and DP178 are potent inhibitorsof HIV infection and fusion, probably by virtue of their ability to formcomplexes with viral gp41 and interfere with its fusogenic process;e.g., during the structural transition of the viral protein from thenative structure to the fusogenic state, the DP178 and DP107 peptidesmay gain access to their respective binding sites on the viral gp41, andexert a disruptive influence. DP107 peptides which demonstrate anti-HIVactivity are described in Applicants' co-pending application Ser. No.08/264,531, filed Jun. 23, 1994, which is incorporated by referenceherein in its entirety.

As shown in the Examples, infra, a truncated recombinant gp41 proteincorresponding to the ectodomain of gp41 containing both DP107 and DP178domains (excluding the fusion peptide, transmembrane region andcytoplasmic domain of gp41) did not inhibit HIV-1 induced fusion.However, when a single mutation was introduced to disrupt thecoiled-coil structure of the DP107 domain—a mutation which results in atotal loss of biological activity of DP107 peptides—the inactiverecombinant protein was transformed to an active inhibitor of HIV-1induced fusion. This transformation may result from liberation of thepotent DP178 domain from a molecular clasp with the leucine zipper,DP107 domain.

For clarity of discussion, the invention will be described primarily forDP178 peptide inhibitors of HIV. However, the principles may beanalogously applied to other viruses, both enveloped and nonenveloped,and to other non-viral organisms.

5.1. DP178 and DP178-Like Peptides

The DP178 peptide (SEQ ID:1) of the invention corresponds to amino acidresidues 638 to 673 of the transmembrane protein gp41 from theHIV-1_(LAI) isolate, and has the 36 amino acid sequence (reading fromamino to carboxy terminus):NH₂—YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF—COOH  (SEQ ID:1)

In addition to the full-length DP178 (SEQ ID:1) 36-mer, the peptides ofthe invention may include truncations of the DP178 (SEQ ID:1) peptidewhich exhibit antifusogenic activity, antiviral activity and/or theability to modulate intracellular processes involving coiled-coilpeptide structures. Truncations of DP178 (SEQ ID:1) peptides maycomprise peptides of between 3 and 36 amino acid residues (i.e.,peptides ranging in size from a tripeptide to a 36-mer polypeptide), asshown in Tables I and IA, below.

Peptide sequences in these tables are listed from amino (left) tocarboxy (right) terminus. “X” may represent an amino group (—NH₂) and“Z” may represent a carboxyl (—COOH) group. Alternatively, “X” mayrepresent a hydrophobic group, including but not limited to carbobenzyl,dansyl, or T-butoxycarbonyl; an acetyl group; a9-fluorenylmethoxy-carbonyl (FMOC) group; or a covalently attachedmacromolecular group, including but not limited to a lipid-fatty acidconjugate, polyethylene glycol, carbohydrate or peptide group. Further,“Z” may represent an amido group; a T-butoxycarbonyl group; or acovalently attached macromolecular group, including but not limited to alipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptidegroup. A preferred “X” or “Z” macromolecular group is a peptide group.TABLE I DP178 (SEQ ID:1) CARBOXY TRUNCATIONS X-YTS-Z X-YTSL-Z X-YTSLI-ZX-YTSLIH-Z X-YTSLIHS-Z X-YTSLIHSL-Z X-YTSLIHSLI-Z X-YTSLIHSLIE-ZX-YTSLIHSLIEE-Z X-YTSLIHSLIEES-Z X-YTSLIHSLIEESQ-Z X-YTSLIHSLIEESQN-ZX-YTSLIHSLIEESQNQ-Z X-YTSLIHSLIEESQNQQ-Z X-YTSLIHSLIEESQNQQE-ZX-YTSLIHSLIEESQNQQEK-Z X-YTSLIHSLIEESQNQQEKN-Z X-YTSLIHSLIEESQNQQEKNE-ZX-YTSLIHSLIEESQNQQEKNEQ-Z X-YTSLIHSLIEESQNQQEKNEQE-ZX-YTSLIHSLIEESQNQQEKNEQEL-Z X-YTSLIHSLIEESQNQQEKNEQELL-ZX-YTSLIHSLIEESQNQQEKNEQELLE-Z X-YTSLIHSLIEESQNQQEKNEQELLEL-ZX-YTSLIHSLIEESQNQQEKNEQELLELD-Z X-YTSLIHSLIEESQNQQEKNEQELLELDK-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKW-Z X-YTSLIHSLIEESQNQQEKNEQELLELDKWA-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWAS-Z X-YTSLIHSLIEESQNQQEKNEQELLELDKWASL-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLW-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWN-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE IA DP178 (SEQ ID:1) AMINO TRUNCATIONS X-NWF-Z X-WNWF-Z X-LWNWF-ZX-SLWNWF-Z X-ASLWNWF-Z X-WASLWNWF-Z X-KWASLWNWF-Z X-DKWASLWNWF-ZX-LDKWASLWNWF-Z X-ELDKWASLWNWF-Z X-LELDKWASLWNWF-Z X-LLELDKWASLWNWF-ZX-ELLELDKWASLWNWF-Z X-QELLELDKWASLWNWF-Z X-EQELLELDKWASLWNWF-ZX-NEQELLELDKWASLWNWF-Z X-KNEQELLELDKWASLWNWF-Z X-EKNEQELLELDKWASLWNWF-ZX-QEKNEQELLELDKWASLWNWF-Z X-QQEKNEQELLELDKWASLWNWF-ZX-NQQEKNEQELLELDKWASLWNWF-Z X-QNQQEKNEQELLELDKWASLWNWF-ZX-SQNQQEKNEQELLELDKWASLWNWF-Z X-ESQNQQEKNEQELLELDKWASLWNWF-ZX-EESQNQQEKNEQELLELDKWASLWNWF-Z X-IEESQNQQEKNEQELLELDKWASLWNWF-ZX-LIEESQNQQEKNEQELLELDKWASLWNWF-Z X-SLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-HSLIEESQNQQEKNEQELLELDKWASLWNWF-Z X-IHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-LIHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-SLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-TSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl group; a macromolecular carriergroup including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.

The peptides of the invention also include DP178-like peptides.“DP178-like”, as used herein, refers, first, to DP178 and DP178truncations which contain one or more amino acid substitutions,insertions and/or deletions. Second, “DP-178-like” refers to peptidesequences identified or recognized by the ALLMOTI5, 107×178×4 and PLZIPsearch motifs described herein, having structural and/or amino acidmotif similarity to DP178. The DP178-like peptides of the invention mayexhibit antifusogenic or antiviral activity, or may exhibit the abilityto modulate intracellular processes involving coiled-coil peptides.Further, such DP178-like peptides may possess additional advantageousfeatures, such as, for example, increased bioavailability, and/orstability, or reduced host immune recognition.

HIV-1 and HIV-2 enveloped proteins are structurally distinct, but thereexists a striking amino acid conservation within the DP178-correspondingregions of HIV-1 and HIV-2. The amino acid conservation is of a periodicnature, suggesting some conservation of structure and/or function.Therefore, one possible class of amino acid substitutions would includethose amino acid changes which are predicted to stabilize the structureof the DP178 peptides of the invention. Utilizing the DP178 and DP178analog sequences described herein, the skilled artisan can readilycompile DP178 consensus sequences and ascertain from these, conservedamino acid residues which would represent preferred amino acidsubstitutions.

The amino acid substitutions may be of a conserved or non-conservednature. Conserved amino acid substitutions consist of replacing one ormore amino acids of the DP178 (SEQ ID:1) peptide sequence with aminoacids of similar charge, size, and/or hydrophobicity characteristics,such as, for example, a glutamic acid (E) to aspartic acid (D) aminoacid substitution. Non-conserved substitutions consist of replacing oneor more amino acids of the DP178 (SEQ ID:1) peptide sequence with aminoacids possessing dissimilar charge, size, and/or hydrophobicitycharacteristics, such as, for example, a glutamic acid (E) to valine (V)substitution.

Amino acid insertions may consist of single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of the DP178 or DP178 truncated peptides, as well asat a position internal to the peptide. Such insertions will generallyrange from 2 to 15 amino acids in length. It is contemplated thatinsertions made at either the carboxy or amino terminus of the peptideof interest may be of a broader size range, with about 2 to about 50amino acids being preferred. One or more such insertions may beintroduced into DP178 (SEQ.ID:1) or DP178 truncations, as long as suchinsertions result in peptides which may still be recognized by the107×178×4, ALLMOTI5 or PLZIP search motifs described herein, or may,alternatively, exhibit antifusogenic or antiviral activity, or exhibitthe ability to modulate intracellular processes involving coiled-coilpeptide structures.

Preferred amino or carboxy terminal insertions are peptides ranging fromabout 2 to about 50 amino acid residues in length, corresponding to gp41protein regions either amino to or carboxy to the actual DP178 gp41amino acid sequence, respectively. Thus, a preferred amino terminal orcarboxy terminal amino acid insertion would contain gp41 amino acidsequences found immediately amino to or carboxy to the DP178 region ofthe gp41 protein.

Deletions of DP178 (SEQ ID:1) or DP178 truncations are also within thescope of the invention. Such deletions consist of the removal of one ormore amino acids from the DP178 or DP178-like peptide sequence, with thelower limit length of the resulting peptide sequence being 4 to 6 aminoacids. Such deletions may involve a single contiguous or greater thanone discrete portion of the peptide sequences. One or more suchdeletions may be introduced into DP178 (SEQ.ID:1) or DP178 truncations,as long as such deletions result in peptides which may still berecognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs describedherein, or may, alternatively, exhibit antifusogenic or antiviralactivity, or exhibit the ability to modulate intracellular processesinvolving coiled-coil peptide structures.

DP178 analogs are further described, below, in Section 5.3.

5.2. DP107 and DP107-Like Peptides

Further, the peptides of the invention include peptides having aminoacid sequences corresponding to DP107 analogs. DP107 is a 38 amino acidpeptide which exhibits potent antiviral activity, and corresponds toresidues 558 to 595 of HIV-1_(LAI) transmembrane (TM) gp41 protein, asshown here:NH₂—NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-COOH  (SEQ ID:25)

In addition to the full-length DP107 (SEQ ID:25) 38-mer, the peptides ofthe invention may include truncations of the DP107 (SEQ ID:25) peptidewhich exhibit antifusogenic activity, antiviral activity and/or theability to modulate intracellular processes involving coiled-coilpeptide structures. Truncations of DP107 (SEQ ID:25) peptides maycomprise peptides of between 3 and 38 amino acid residues (i.e.,peptides ranging in size from a tripeptide to a 38-mer polypeptide), asshown in Tables II and IIA, below. Peptide sequences in these tables arelisted from amino (left) to carboxy (right) terminus. “X” may representan amino group (—NH₂) and “Z” may represent a carboxyl (—COOH) group.Alternatively, “X” may represent a hydrophobic group, including but notlimited to carbobenzyl, dansyl, or T-butoxycarbonyl; an acetyl group; a9-fluorenylmethoxy-carbonyl (FMOC) group; or a covalently attachedmacromolecular group, including but not limited to a lipid-fatty acidconjugate, polyethylene glycol, carbohydrate or peptide group. Further,“Z” may represent an amido group; a T-butoxycarbonyl group; or acovalently attached macromolecular group, including but not limited to alipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptidegroup. A preferred “X” or “Z” macromolecular group is a peptide group.TABLE II DP107 (SEQ ID:25) CARBOXY TRUNCATIONS X-NNL-Z X-NNLL-ZX-NNLLR-Z X-NNLLRA-Z X-NNLLRAI-Z X-NNLLRAIE-Z X-NNLLRAIEA-ZX-NNLLRAIEAQ-Z X-NNLLRAIEAQQ-Z X-NNLLRAIEAQQH-Z X-NNLLRAIEAQQHL-ZX-NNLLRAIEAQQHLL-Z X-NNLLRAIEAQQHLLQ-Z X-NNLLRAIEAQQHLLQL-ZX-NNLLRAIEAQQHLLQLT-Z X-NNLLRAIEAQQHLLQLTV-Z X-NNLLRAIEAQQHLLQLTVW-ZX-NNLLRAIEAQQHLLQLTVWQ-Z X-NNLLRAIEAQQHLLQLTVWQI-ZX-NNLLRAIEAQQHLLQLTVWQIK-Z X-NNLLRAIEAQQHLLQLTVWQIKQ-ZX-NNLLRAIEAQQHLLQLTVWQIKQL-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQ-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQA-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQAR-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARI-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQARIL-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILA-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQARILAV-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVE-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVER-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERY-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYL-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLK-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKD-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE IIA DP178 (SEQ ID:25) AMINO TRUNCATIONS X-KDQ-Z X-LKDQ-Z X-YLKDQ-ZX-RYLKDQ-Z X-ERYLKDQ-Z X-VERYLKDQ-Z X-AVERYLKDQ-Z X-LAVERYLKDQ-ZX-ILAVERYLKDQ-Z X-RILAVERYLKDQ-Z X-ARILAVERYLKDQ-Z X-QARILAVERYLKDQ-ZX-LQARILAVERYLKDQ-Z X-QLQARILAVERYLKDQ-Z X-KQLQARILAVERYLKDQ-ZX-IKQLQARILAVERYLKDQ-Z X-QIKQLQARILAVERYLKDQ-Z X-WQIKQLQARILAVERYLKDQ-ZX-VWQIKQLQARILAVERYLKDQ-Z X-TVWQIKQLQARILAVERYLKDQ-ZX-LTVWQIKQLQARILAVERYLKDQ-Z X-QLTVWQIKQLQARILAVERYLKDQ-ZX-LQLTVWQIKQLQARILAVERYLKDQ-Z X-LLQLTVWQIKQLQARILAVERYLKDQ-ZX-HLLQLTVWQIKQLQARILAVERYLKDQ-Z X-QHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-QQHLLQLTVWQIKQLQARILAVERYLKDQ-Z X-AQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-EAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z X-IEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-AIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-RAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-LRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-LLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-NLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl group; a macromolecular carriergroup including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.

The peptides of the invention also include DP107-like peptides.“DP107-like”, as used herein, refers, first, to DP107 and DP107truncations which contain one or more amino acid substitutions,insertions and/or deletions. Second, “DP-107-like” refers to peptidesequences identified or recognized by the ALLMOTI5, 107×178×4 and PLZIPsearch motifs described herein, having structural and/or amino acidmotif similarity to DP107. The DP107-like peptides of the invention mayexhibit antifusogenic or antiviral activity, or may exhibit the abilityto modulate intracellular processes involving coiled-coil peptides.Further, such DP107-like peptides may possess additional advantageousfeatures, such as, for example, increased bioavailability, and/orstability, 1 or reduced host immune recognition.

HIV-1 and HIV-2 enveloped proteins are structurally distinct, but thereexists a striking amino acid conservation within the DP107-correspondingregions of HIV-1 and HIV-2. The amino acid conservation is of a periodicnature, suggesting some conservation of structure and/or function.Therefore, one possible class of amino acid substitutions would includethose amino acid changes which are predicted to stabilize the structureof the DP107 peptides of the invention. Utilizing the DP107 and DP107analog sequences described herein, the skilled artisan can readilycompile DP107 consensus sequences and ascertain from these, conservedamino acid residues which would represent preferred amino acidsubstitutions.

The amino acid substitutions may be of a conserved or non-conservednature. Conserved amino acid substitutions consist of replacing one ormore amino acids of the DP107 (SEQ ID:25) peptide sequence with aminoacids of similar charge, size, and/or hydrophobicity characteristics,such as, for example, a glutamic acid (E) to aspartic acid (D) aminoacid substitution. Non-conserved substitutions consist of replacing oneor more amino acids of the DP107 (SEQ ID:25) peptide sequence with aminoacids possessing dissimilar charge, size, and/or hydrophobicitycharacteristics, such as, for example, a glutamic acid (E) to valine (V)substitution.

Amino acid insertions may consist of single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of the DP107 or DP107 truncated peptides, as well asat a position internal to the peptide. Such insertions will generallyrange from 2 to 15 amino acids in length. It is contemplated thatinsertions made at either the carboxy or amino terminus of the peptideof interest may be of a broader size range, with about 2 to about 50amino acids being preferred. One or more such insertions may beintroduced into DP107 (SEQ.ID:25) or DP107 truncations, as long as suchinsertions result in peptides which may still be recognized by the107×178×4, ALLMOTI5 or PLZIP search motifs described herein, or may,alternatively, exhibit antifusogenic or antiviral activity, or exhibitthe ability to modulate intracellular processes involving coiled-coilpeptide structures.

Preferred amino or carboxy terminal insertions are peptides ranging fromabout 2 to about 50 amino acid residues in length, corresponding to gp41protein regions either amino to or carboxy to the actual DP107 gp41amino acid sequence, respectively. Thus, a preferred amino terminal orcarboxy terminal amino acid insertion would contain gp41 amino acidsequences found immediately amino to or carboxy to the DP107 region ofthe gp41 protein.

Deletions of DP107 (SEQ ID:25) or DP178 truncations are also within thescope of the invention. Such deletions consist of the removal of one ormore amino acids from the DP107 or DP107-like peptide sequence, with thelower limit length of the resulting peptide sequence being 4 to 6 aminoacids. Such deletions may involve a single contiguous or greater thanone discrete portion of the peptide sequences. One or more suchdeletions may be introduced into DP107 (SEQ.ID:25) or DP107 truncations,as long as such deletions result in peptides which may still berecognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs describedherein, or may, alternatively, exhibit antifusogenic or antiviralactivity, or exhibit the ability to modulate intracellular processesinvolving coiled-coil peptide structures.

DP107 and DP107 truncations are more fully described in Applicants'co-pending U.S. patent application Ser. No. 08/374,666, filed Jan. 27,1995, and which is incorporated herein by reference in its entirety.DP107 analogs are further described, below, in Section 5.3.

5.3. DP107 and DP178 Analogs

Peptides corresponding to analogs of the DP178, DP178 truncations, DP107and DP107 truncation sequences of the invention, described, above, inSections 5.1 and 5.2 may be found in other viruses, including, forexample, non-HIV-1_(LAI) enveloped viruses, non-enveloped viruses andother non-viral organisms.

The term “analog”, as used herein, refers to a peptide which isrecognized or identified via the 107×178×4, ALLMOTI5 and/or PLZIP searchstrategies discussed below. Further, such peptides may exhibitantifusogenic capability, antiviral activity, or the ability to modulateintracellular processes involving coiled-coil structures.

Such DP178 and DP107 analogs may, for example, correspond to peptidesequences present in TM proteins of enveloped viruses and may,additionally correspond to peptide sequences present in non envelopedand non-viral organisms. Such peptides may exhibit antifusogenicactivity, antiviral activity, most particularly antiviral activity whichis specific to the virus in which their native sequences are found, ormay exhibit an ability to modulate intracellular processes involvingcoiled-coil peptide structures.

DP178 analogs are peptides whose amino acid sequences are comprised ofthe amino acid sequences of peptide regions of, for example, other(i.e., other than HIV-1_(LAI)) viruses that correspond to the gp41peptide region from which DP178 (SEQ ID:1) was derived. Such viruses mayinclude, but are not limited to, other HIV-1 isolates and HIV-2isolates.

DP178 analogs derived from the corresponding gp41 peptide region ofother (i.e., non HIV-1_(LAI)) HIV-1 isolates may include, for example,peptide sequences as shown below. (DP-185; SEQ ID:3)NH₂-YTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF-COOH; (SEQ ID:4)NH₂-YTGIIYNLLEESQNQQEKNEQELLELDKWANLWNWF-COOH; (SEQ ID:5)NH₂-YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF-COOH.SEQ ID:3 (DP-185), SEQ ID:4, and SEQ ID:5 are derived from HIV-1_(SF2),HIV-1_(RF), and HIV-1_(MN) isolates, respectively. Underlined amino acidresidues refer to those residues that differ from the correspondingposition in the DP178 (SEQ ID:1) peptide. One such DP178 analog, DP-185(SEQ ID:3), is described in the Example presented in Section 6, below,where it is demonstrated that DP-185 (SEQ ID:3) exhibits antiviralactivity. The DP178 analogs of the invention may also includetruncations, as described above. Further, the analogs of the inventionmodifications such those described for DP178 analogs in Section 5.1.,above. It is preferred that the DP178 analogs of the invention representpeptides whose amino acid sequences correspond to the DP178 region ofthe gp41 protein, it is also contemplated that the peptides of theinvention may, additionally, include amino sequences, ranging from about2 to about 50 amino acid residues in length, corresponding to gp41protein regions either amino to or carboxy to the actual DP178 aminoacid sequence.

Striking similarities, as shown in FIG. 1, exist within the regions ofHIV-1 and HIV-2 isolates which correspond to the DP178 sequence. A DP178analog derived from the HIV-2_(NIHz) isolate has the 36 amino acidsequence (reading from amino to carboxy terminus): (SEQ ID: 7)NH₂-LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-COOH

Table III and Table IV show some possible truncations of theHIV-2_(NIHz) DP178 analog, which may comprise peptides of between 3 and36 amino acid residues (i.e., peptides ranging in size from a tripeptideto a 36-mer polypeptide). Peptide sequences in these tables are listedfrom amino (left) to carboxy (right) terminus. “X” may represent anamino group (—NH₂) and “Z” may represent a carboxyl (—COOH) group.Alternatively, “X” may represent a hydrophobic group, including but notlimited to carbobenzyl, dansyl, or T-butoxycarbonyl; an acetyl group; a9-fluorenylmethoxy-carbonyl (FMOC) group; or a covalently attachedmacromolecular group, including but not limited to a lipid-fatty acidconjugate, polyethylene glycol, carbohydrate or peptide group. Further,“Z” may represent an amido group; a T-butoxycarbonyl group; or acovalently attached macromolecular group, including but not limited to alipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptidegroup. A preferred “X” or “Z” macromolecular group is a peptide group.TABLE III HIV-2_(NIHZ) DP178 analog carboxy truncations. X-LEA-ZX-LEAN-Z X-LEANI-Z X-LEANIS-Z X-LEANISQ-Z X-LEANISQS-Z X-LEANISQSL-ZX-LEANISQSLE-Z X-LEANISQSLEQ-Z X-LEANISQSLEQA-Z X-LEANISQSLEQAQ-ZX-LEANISQSLEQAQI-Z X-LEANISQSLEQAQIQ-Z X-LEANISQSLEQAQIQQ-ZX-LEANISQSLEQAQIQQE-Z X-LEANISQSLEQAQIQQEK-Z X-LEANISQSLEQAQIQQEKN-ZX-LEANISQSLEQAQIQQEKNM-Z X-LEANISQSLEQAQIQQEKNMY-ZX-LEANISQSLEQAQIQQEKNMYE-Z X-LEANISQSLEQAQIQQEKNMYEL-ZX-LEANISQSLEQAQIQQEKNMYELQ-Z X-LEANISQSLEQAQIQQEKNMYELQK-ZX-LEANISQSLEQAQIQQEKNMYELQKL-Z X-LEANISQSLEQAQIQQEKNMYELQKLN-ZX-LEANISQSLEQAQIQQEKNMYELQKLNS-Z X-LEANISQSLEQAQIQQEKNMYELQKLNSW-ZX-LEANISQSLEQAQIQQEKNMYELQKLNSWD-Z X-LEANISQSLEQAQIQQEKNMYELQKLNSWDV-ZX-LEANISQSLEQAQIQQEKNMYELQKLNSWDVF-ZX-LEANISQSLEQAQIQQEKNMYELQKLNSWDVFT-ZX-LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTN-ZX-LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNW-ZX-LEANISQSLEQAQIQQEKNMYELQKLMSWDVFTNWL-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE IV HIV-2_(NIHZ) DP178 analog amino truncations. X-NWL-Z X-TNWL-ZX-FTNWL-Z X-VFTNWL-Z X-DVFTNWL-Z X-WDVFTNWL-Z X-SWDVFTNWL-ZX-NSWDVFTNWL-Z X-LNSWDVFTNWL-Z X-KLNSWDVFTNWL-Z X-QKLNSWDVFTNWL-zX-LQKLNSWDVFTNWL-Z X-ELQKLNSWDVFTNWL-Z X-YELQKLNSWDVFTNWL-ZX-MYELQKLNSWDVFTNWL-Z X-NMYELQKLNSWDVFTNWL-Z X-KNMYELQKLNSWDVFTNWL-ZX-EKNMYELQKLNSWDVFTNWL-Z X-QEKNMYELQKLNSWDVFTNWL-ZX-QQEKNMYELQKLNSWDVFTNWL-Z X-IQQEKNMYELQKLNSWDVFTNWL-ZX-QIQQEKNMYELQKLNSWDVFTNWL-Z X-AQIQQEKNMYELQKLNSWDVFTNWL-ZX-QAQIQQEKNMYELQKLNSWDVFTNWL-Z X-EQAQIQQEKNMYELQKLNSWDVFTNWL-ZX-LEQAQIQQEKNMYELQKLNSWDVFTNWL-Z X-SLEQAQIQQEKNMYELQKLNSWDVFTNWL-ZX-QSLEQAQIQQEKNMYELQKLNSWDVFTNWL-Z X-SQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-ZX-ISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-ZX-NISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-ZX-ANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-ZX-EANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-ZX-LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.

DP178 and DP107 analogs are recognized or identified, for example, byutilizing one or more of the 107×178×4, ALLMOTI5 or PLZIPcomputer-assisted search strategies described and demonstrated, below,in the Examples presented in Sections 9 through 16 and 19 through 25.The search strategy identifies additional peptide regions which arepredicted to have structural and/or amino acid sequence features similarto those of DP107 and/or DP178.

The search strategies are described fully, below, in the Examplepresented in Section 9. While this search strategy is based, in part, ona primary amino acid motif deduced from DP107 and DP178, it is not basedsolely on searching for primary amino acid sequence homologies, as suchprotein sequence homologies exist within, but not between major groupsof viruses. For example, primary amino acid sequence homology is highwithin the TM protein of different strains of HIV-1 or within the TMprotein of different isolates of simian immunodeficiency virus (SIV).Primary amino acid sequence homology between HIV-1 and SIV, however, islow enough so as not to be useful. It is not possible, therefore, tofind peptide regions similar to DP107 or DP178 within other viruses, orwithin non-viral organisms, whether structurally, or otherwise, based onprimary sequence homology, alone.

Further, while it would be potentially useful to identify primarysequence arrangements of amino acids based on, for example, the physicalchemical characteristics of different classes of amino acids rather thanbased on the specific amino acids themselves, such search strategieshave, until now, proven inadequate. For example, a computer algorithmdesigned by Lupas et al. to identify coiled-coil propensities of regionswithin proteins (Lupas, A., et al., 1991 Science 252:1162-1164) isinadequate for identifying protein regions analogous to DP107 or DP178.

Specifically, analysis of HIV-1 gp160 (containing both gp120 and gp41)using the Lupas algorithm does not identify the coiled-coil regionwithin DP107. It does, however, identify a region within DP178 beginningeight amino acids N-terminal to the start of DP178 and ending eightamino acids from the C-terminus. The DP107 peptide has been shownexperimentally to form a stable coiled coil. A search based on the Lupassearch algorithm, therefore, would not have identified the DP107coiled-coil region. Conversely, the Lupas algorithm identified the DP178region as a potential coiled-coil motif. However, the peptide derivedfrom the DP178 region failed to form a coiled coil in solution.

A possible explanation for the inability of the Lupas search algorithmto accurately identify coiled-coil sequences within the HIV-1 TM, isthat the Lupas algorithm is based on the structure of coiled coils fromproteins that are not structurally or functionally similar to the TMproteins of viruses, antiviral peptides (e.g. DP107 and DP178) of whichare an object of this invention.

The computer search strategy of the invention, as demonstrated in theExamples presented below, in Sections 9 through 16 and 19 through 25,successfully identifies regions of proteins similar to DP107 or DP178.This search strategy was designed to be used with acommercially-available sequence database package, preferably PC/Gene.

A series of search motifs, the 107×178×4, ALLMOTI5 and PLZIP motifs,were designed and engineered to range in stringency from strict tobroad, as discussed in this Section and in Section 9, with 107×178×4being preferred. The sequences identified via such search motifs, suchas those listed in Tables V-XIV, below, potentially exhibitantifusogenic, such as antiviral, activity, may additionally be usefulin the identification of antifusogenic, such as antiviral, compounds,and are intended to be within the scope of the invention.

Coiled-coiled sequences are thought to consist of heptad amino acidrepeats. For ease of description, the amino acid positions within theheptad repeats are sometimes referred to as A through G, with the firstposition being A, the second B, etc. The motifs used to identifyDP107-like and DP178-like sequences herein are designed to specificallysearch for and identify such heptad repeats. In the descriptions of eachof the motifs described, below, amino acids enclosed by brackets, i.e.,[ ], designate the only amino acid residues that are acceptable at thegiven position, while amino acids enclosed by braces, i.e., { },designate the only amino acids which are unacceptable at the givenheptad position. When a set of bracketed or braced amino acids isfollowed by a number in parentheses i.e., ( ), it refers to the numberof subsequent amino acid positions for which the designated set of aminoacids hold, e.g, a (2) means “for the next two heptad amino acidpositions”.

The ALLMOTI5 is written as follows:

{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-

{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-

{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-

{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-

{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-

Translating this motif, it would read: “at the first (A) position of theheptad, any amino acid residue except C, D, G, H, or P is acceptable, atthe next two (B,C) amino acid positions, any amino acid residue exceptC, F, or P is acceptable, at the fourth heptad position (D), any aminoacid residue except C, D, G, H, or P is acceptable, at the next three(E, F, G) amino acid positions, any amino acid residue except C, F, or Pis acceptable. This motif is designed to search for five consecutiveheptad repeats (thus the repeat of the first line five times), meaningthat it searches for 35-mer sized peptides. It may also be designed tosearch for 28-mers, by only repeating the initial motif four times. Withrespect to the ALLMOTI5 motif, a 35-mer search is preferred. Those viral(non-bacteriophage) sequences identified via such an ALLMOTI5 motif arelisted in Table V, below, at the end of this Section. The viralsequences listed in Table V potentially exhibit antiviral activity, maybe useful in the the identification of antiviral compounds, and areintended to be within the scope of the invention. In those instanceswherein a single gene exhibits greater than one sequence recognized bythe ALLMOTI5 search motif, the amino a cid residue numbers of thesesequences are listed under “Area 2”, Area 3”, etc. This convention isused for each of the Tables listed, below, at the end of this Section.

The 107×178×4 motif is written as follows:

[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY]-{CFMP}(3)-

[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY]-{CFMP}(3)-

[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY]-{CFMP}(3)-

[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY)-{CFMP}(3)-

Translating this motif, it would read: “at the first (A) position of theheptad, only amino acid residue E, F, I, K, L, N, Q, S, T, V, W, or Y isacceptable, at the next two (B,C) amino acid positions, any amino acidresidue except C, F, M or P is acceptable, at the fourth position (D),only amino acid residue E, F, I, K, L, N, Q, S, T, V, W, or Y isacceptable, at the next three (E, F, G) amino acid positions, any aminoacid residue except C, F, M or P is acceptable. This motif is designedto search for four consecutive heptad repeats (thus the repeat of thefirst line four times), meaning that it searches for 28-mer sizedpeptides. It may also be designed to search for 35-mers, by repeatingthe initial motif five times. With respect to the 107×178×4 motif, a28-mer search is preferred.

Those viral (non-bacteriophage) sequences identified via such a107×178×4 motif are listed in Table VI, below, at the end of thisSection, with those viral (non-bacteriophage) sequences listed in TableVII, below at the end of this Section, being preferred.

The 107×178×4 search motif was also utilized to identify non-viralprocaryotic protein sequences, as listed in Table VIII, below, at theend of this Section. Further, this search motif was used to reveal anumber of human proteins. The results of this human protein 107×178×4search is listed in Table IX, below, at the end of this Section. Thesequences listed in Tables VIII and IX, therefore, reveal peptides whichmay be useful as antifusogenic compounds or in the identification ofantifusogenic compounds, and are intended to be within the scope of theinvention.

The PLZIP series of motifs are as listed in FIG. 19. These motifs aredesigned to identify leucine zipper coiled-coil like heptads wherein atleast one proline residue is present at some predefined distanceN-terminal to the repeat. These PLZIP motifs find regions of proteinswith similarities to HIV-1 DP178 generally located just N-terminal tothe transmembrane anchor. These motifs may be translated according tothe same convention described above. Each line depicted in FIG. 19represents a single, complete search motif. “X” in these motifs refersto any amino acid residue. In instances wherein a motif contains twonumbers within parentheses, this refers to a variable number of aminoacid residues. For example, X (1,12) is translated to “the next one totwelve amino acid residues, inclusive, may be any amino acid”.

Tables X through XIV, below, at the end of this Section, list sequencesidentified via searches conducted with such PLZIP motifs. Specifically,Table X lists viral sequences identified via PCTLZIP, P1CTLZIP andP2CTLZIP search motifs, Table XI lists viral sequences identified viaP3CTLZIP, P4CTLZIP, P5CTLZIP and P6CTLZIP search motifs, Table XII lstsviral sequences identified via P7CTLZIP, P8CTLZIP and P9CTLZIP searchmotifs, Table XIII lists viral sequences identified via P12LZIPCsearches and Table XIV lists viral sequences identified via P23TLZIPCsearch motifs The viral sequences listed in these tables representpeptides which potentially exhibit antiviral activity, may be useful inthe identification of antiviral compounds, and are intended to be withinthe scope of the invention.

The Examples presented in Sections 17, 18, 26 and 27 below, demonstratethat viral sequences identified via the motif searches described hereinidentify substantial antiviral characteristics. Specifically, theExample presented in Section 17 describes peptides with anti-respiratorysyncytial virus activity, the Example presented in Section 18 describespeptides with anti-parainfluenza virus activity, the Example presentedin Section 26 describes peptides with anti-measles virus activity andthe Example presented in Section 27 describes peptides with anti-simianimmunodeficiency virus activity.

The DP107 and DP178 analogs may, further, contain any of the additionalgroups described for DP178, above, in Section 5.1. For example, thesepeptides may include any of the additional amino-terminal groups asdescribed above for “X” groups, and may also include any of thecarboxy-terminal groups as described, above, for “Z” groups.

Additionally, truncations of the identified DP107 and DP178 peptides areamong the peptides of the invention. Further, such DP107 and DP178analogs and DP107/DP178 analog truncations may exhibit one or more aminoacid substitutions, insertion, and/or deletions. The DP178 analog aminoacid substitutions, insertions and deletions, are as described, above,for DP178-like peptides in Section 5.1. The DP-107 analog amino acidsubstitutions, insertions and deletions are also as described, above,for DP107-like peptides in Section 5.2.

Tables XV through XXII, below, present representative examples of suchDP107/DP178 truncations. Specifically, Table XV presents RespiratorySyncytial Virus F1 region DP107 analog carboxy truncations, Table XVIpresents Respiratory Syncytial Virus F1 region DP107 analog aminotruncations, Table XVII presents Respiratory Syncytial Virus F1 regionDP178 analog carboxy truncations, Table XVIII presents RespiratorySyncytial Virus F1 region DP178 analog amino truncations, Table XIXpresents Human Parainfluenza Virus 3 F1 region DP178 analog carboxytruncations, Table XX presents Human Parainfluenza Virus 3 F1 regionDP178 analog amino truncations, Table XXI presents Human ParainfluenzaVirus 3 F1 region DP107 analog carboxy truncations and Table XXIIpresents Human Parainfluenza Virus 3 F1 region DP107 analog aminotruncations. Further, Table XXIII, below, presents DP107/DP178 analogsand analog truncations which exhibit substantial antiviral activity.These antiviral peptides are grouped according to the specific viruswhich they inhibit, including respiratory syncytial virus, humanparainfluenza virus 3, simian immunodeficiency virus and measles virus.LENGTHY TABLE REFERENCED HERE US20070037141A1-20070215-T00001 Pleaserefer to the end of the specification for access instructions. LENGTHYTABLE REFERENCED HERE US20070037141A1-20070215-T00002 Please refer tothe end of the specification for access instructions. LENGTHY TABLEREFERENCED HERE US20070037141A1-20070215-T00003 Please refer to the endof the specification for access instructions. LENGTHY TABLE REFERENCEDHERE US20070037141A1-20070215-T00004 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070037141A1-20070215-T00005 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070037141A1-20070215-T00006 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070037141A1-20070215-T00007 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070037141A1-20070215-T00008 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070037141A1-20070215-T00009 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070037141A1-20070215-T00010 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070037141A1-20070215-T00011 Please refer to the end of thespecification for access instructions.

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XVI RESPIRATORY SYNCYTIAL VIRUS F2 DP178/DP107 REGION ANALOG AMINOTRUNCATIONS                                             X-QST-Z                                           X-MQST-Z                                          X-LMQST-Z                                         X-LLMQST-Z                                        X-QLLMQST-Z                                       X-LQLLMQST-Z                                      X-ELQLLMQST-Z                                     X-TELQLLMQST-Z                                    X-VTELQLLMQST-Z                                   X-AVTELQLLMQST-Z                                  X-NAVTELQLLMQST-Z                                 X-KNAVTELQLLMQST-Z                                X-YKNAVTELQLLMQST-Z                               X-KYKNAVTELQLLMQST-Z                              X-DKYKNAVTELQLLMQST-Z                             X-LDKYKNAVTELQLLMQST-Z                            X-ELDKYKNAVTELQLLMQST-Z                           X-QELDKYKNAVTELQLLMQST-Z                          X-KQELDKYKNAVTELQLLMQST-Z                         X-IKQELDKYKNAVTELQLLMQST-Z                        X-LIKQELDKYKNAVTELQLLMQST-Z                       X-KLIKQELDKYKNAVTELQLLMQST-Z                      X-VKLIKQELDKYKNAVTELQLLMQST-Z                     X-KVKLIKQELDKYKNAVTELQLLMQST-Z                    X-AKVKLIKQELDKYKNAVTELQLLMQST-Z                   X-DAKVKLIKQELDKYKNAVTELQLLMQST-Z                  X-TDAKVKLIKQELDKYKNAVTELQLLMQST-Z                 X-GTDAKVKLIKQELDKYKNAVTELQLLMQST-Z                X-NGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z               X-CNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z              X-KCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z             X-NKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z           X-KENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z          X-IKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z         X-NIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z        X-SNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z       X-LSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z      X-ELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z     X-IELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z    X-TIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z   X-ITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z  X-VITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z X-SVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-ZX-TSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XVII RESPIRATORY SYNCYTIAL VIRUS F1 DP178 REGION ANALOG CARBOXYTRUNCATIONS X-FYD-Z X-FYDP-Z X-FYDPL-Z X-FYDPLV-Z X-FYDPLVF-ZX-FYDPLVFP-Z X-FYDPLVFPS-Z X-FYDPLVFPSD-Z X-FYDPLVFPSDE-ZX-FYDPLVFPSDEF-Z X-FYDPLVFPSDEFD-Z X-FYDPLVFPSDEFDA-ZX-FYDPLVFPSDEFDAS-Z X-FYDPLVFPSDEFDASI-Z X-FYDPLVFPSDEFDASIS-ZX-FYDPLVFPSDEFDASISQ-Z X-FYDPLVFPSDEFDASISQV-Z X-FYDPLVFPSDEFDASISQVN-ZX-FYDPLVFPSDEFDASISQVNE-Z X-FYDPLVFPSDEFDASISQVNEK-ZX-FYDPLVFPSDEFDASISQVNEKI-Z X-FYDPLVFPSDEFDASISQVNEKIN-ZX-FYDPLVFPSDEFDASISQVNEKINQ-Z X-FYDPLVFPSDEFDASISQVNEKINQS-ZX-FYDPLVFPSDEFDASISQVNEKINQSL-Z X-FYDPLVFPSDEFDASISQVNEKINQSLA-ZX-FYDPLVFPSDEFDASISQVNEKINQSLAF-Z X-FYDPLVFPSDEFDASISQVNEKINQSLAFI-ZX-FYDPLVFPSDEFDASISQVNEKINQSLAFIR-Z X-FYDPLVFPSDEFDASISQVNEKINQSLAFIRK-ZX-FYDPLVFPSDEFDASISQVNEKINQSLAFIRKS-ZX-FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSD-ZX-FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE-ZX-FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL-ZX-FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XVIII RESPIRATORY SYNCYTIAL VIRUS F1 DP178 REGION ANALOG AMINOTRUNCATIONS X-DELL-Z X-SDELL-Z X-KSDELL-Z X-RKSDELL-Z X-IRKSDELL-ZX-FIRKSDELL-Z X-AFIRKSDELL-Z X-LAFIRKSDELL-Z X-SLAFIRKSDELL-ZX-QSLAFIRKSDELL-Z X-NQSLAFIRKSDELL-Z X-INQSLAFIRKSDELL-ZX-KINQSLAFIRKSDELL-Z X-EKINQSLAFIRKSDELL-Z X-NEKINQSLAFIRKSDELL-ZX-VNEKINQSLAFIRKSDELL-Z X-QVNEKINQSLAFIRKSDELL-ZX-SQVNEKINQSLAFIRKSDELL-Z X-ISQVNEKINQSLAFIRKSDELL-ZX-SISQVNEKINQSLAFIRKSDELL-Z X-ASISQVNEKINQSLAFIRKSDELL-ZX-DASISQVNEKINQSLAFIRKSDELL-Z X-FDASISQVNEKINQSLAFIRKSDELL-ZX-EFDASISQVNEKINQSLAFIRKSDELL-Z X-DEFDASISQVNEKINQSLAFIRKSDELL-ZX-SDEFDASISQVNEKINQSLAFIRKSDELL-Z X-PSDEFDASISQVNEKINQSLAFIRKSDELL-ZX-FPSDEFDASISQVNEKINQSLAFIRKSDELL-Z X-VFPSDEFDASISQVNEKINQSLAFIRKSDELL-ZX-LVFPSDEFDASISQVNEKINQSLAFIRKSDELL-ZX-PLVFPSDEFDASISQVNEKINQSLAFIRKSDELL-ZX-DPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL-ZX-YDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XIX HUMAN PARAINFLUENZA VIRUS 3 F1 REGION DP178 ANALOG CARBOXYTRUNCATIONS X-ITL-Z X-ITLN-Z X-ITLNN-Z X-ITLNNS-Z X-ITLNNSV-ZX-ITLNNSVA-Z X-ITLNNSVAL-Z X-ITLNNSVALD-Z X-ITLNNSVALDP-ZX-ITLNNSVALDPI-Z X-ITLNNSVALDPID-Z X-ITLNNSVALDPIDI-ZX-ITLNNSVALDPIDIS-Z X-ITLNNSVALDPIDISI-Z X-ITLNNSVALDPIDISIE-ZX-ITLNNSVALDPIDISIEL-Z X-ITLNNSVALDPIDISIELN-Z X-ITLNNSVALDPIDISIELNK-ZX-ITLNNSVALDPIDISIELNKA-Z X-ITLNNSVALDPIDISIELNKAK-ZX-ITLNNSVALDPIDISIELNKAKS-Z X-ITLNNSVALDPIDISIELNKAKSD-ZX-ITLNNSVALDPIDISIELNKAKSDL-Z X-ITLNNSVALDPIDISIELNKAKSDLE-ZX-ITLNNSVALDPIDISIELNKAKSDLEE-Z X-ITLNNSVALDPIDISIELNKAKSDLEES-ZX-ITLNNSVALDPIDISIELNKAKSDLEESK-Z X-ITLNNSVALDPIDISIELNKAKSDLEESKE-ZX-ITLNNSVALDPIDISIELNKAKSDLEESKEW-Z X-ITLNNSVALDPIDISIELNKAKSDLEESKEWI-ZX-ITLNNSVALDPIDISIELNKAKSDLEESKEWIR-ZX-ITLNNSVALDPIDISIELNKAKSDLEESKEWIRR-ZX-ITLNNSVALDPIDISIELNKAKSDLEESKEWIRRS-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XX HUMAN PARAINFLUENZA VIRUS 3 F1 REGION DP178 ANALOG AMINOTRUNCATIONS X-RRS-Z X-IRRS-Z X-WIRRS-Z X-EWIRRS-Z X-KEWIRRS-ZX-SKEWIRRS-Z X-ESKEWIRRS-Z X-EESKEWIRRS-Z X-LEESKEWIRRS-ZX-DLEESKEWIRRS-Z X-SDLEESKEWIRRS-Z X-KSDLEESKEWIRRS-ZX-AKSDLEESKEWIRRS-Z X-KAKSDLEESKEWIRRS-Z X-NKAKSDLEESKEWIRRS-ZX-LNKAKSDLEESKEWIRRS-Z X-ELNKAKSDLEESKEWIRRS-Z X-IELNKAKSDLEESKEWIRRS-ZX-SIELNKAKSDLEESKEWIRRS-Z X-ISIELNKAKSDLEESKEWIRRS-ZX-DISIELNKAKSDLEESKEWIRRS-Z X-IDISIELNKAKSDLEESKEWIRRS-ZX-PIDISIELNKAKSDLEESKEWIRRS-Z X-DPIDISIELNKAKSDLEESKEWIRRS-ZX-LDPIDISIELNKAKSDLEESKEWIRRS-Z X-ALDPIDISIELNKAKSDLEESKEWIRRS-ZX-VALDPIDISIELNKAKSDLEESKEWIRRS-Z X-SVALDPIDISIELNKAKSDLEESKEWIRRS-ZX-NSVALDPIDISIELNKAKSDLEESKEWIRRS-Z X-NNSVALDPIDISIELNKAKSDLEESKEWIRRS-ZX-LNNSVALDPIDISIELNKAKSDLEESKEWIRRS-ZX-TLNNSVALDPIDISIELNKAKSDLEESKEWIRRS-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XXI HUMAN PARAINFLUENZA VIRUS 3 F1 REGION DP107 ANALOG CARBOXYTRUNCATIONS X-ALG-Z X-ALGV-Z X-ALGVA-Z X-ALGVAT-Z X-ALGVATS-ZX-ALGVATSA-Z X-ALGVATSAQ-Z X-ALGVATSAQI-Z X-ALGVATSAQIT-ZX-ALGVATSAQITA-Z X-ALGVATSAQITAA-Z X-ALGVATSAQITAAV-ZX-ALGVATSAQITAAVA-Z X-ALGVATSAQITAAVAL-Z X-ALGVATSAQITAAVALV-ZX-ALGVATSAQITAAVALVE-Z X-ALGVATSAQITAAVALVEA-Z X-ALGVATSAQITAAVALVEAK-ZX-ALGVATSAQITAAVALVEAKQ-Z X-ALGVATSAQITAAVALVEAKQA-ZX-ALGVATSAQITAAVALVEAKQAR-Z X-ALGVATSAQITAAVALVEAKQARS-ZX-ALGVATSAQITAAVALVEAKQARSD-Z X-ALGVATSAQITAAVALVEAKQARSDI-ZX-ALGVATSAQITAAVALVEAKQARSDIE-Z X-ALGVATSAQITAAVALVEAKQARSDIEK-ZX-ALGVATSAQITAAVALVEAKQARSDIEKL-Z X-ALGVATSAQITAAVALVEAKQARSDIEKLK-ZX-ALGVATSAQITAAVALVEAKQARSDIEKLKE-Z X-ALGVATSAQITAAVALVEAKQARSDIEKLKEA-ZX-ALGVATSAQITAAVALVEAKQARSDIEKLKEAI-ZX-ALGVATSAQITAAVALVEAKQARSDIEKLKEAIR-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XXII HUMAN PARAINFLUENZA VIRUS 3 F1 REGION DP107 ANALOG AMINOTRUNCATIONS X-IRD-Z X-AIRD-Z X-EAIRD-Z X-KEAIRD-Z X-LKEAIRD-ZX-KLKEAIRD-Z X-EKLKEAIRD-Z X-IEKLKEAIRD-Z X-DIEKLKEAIRD-ZX-SDIEKLKEAIRD-Z X-RSDIEKLKEAIRD-Z X-ARSDIEKLKEAIRD-ZX-QARSDIEKLKEAIRD-Z X-KQARSDIEKLKEAIRD-Z X-AKQARSDIEKLKEAIRD-ZX-EAKQARSDIEKLKEAIRD-Z X-VEAKQARSDIEKLKEAIRD-Z X-LVEAKQARSDIEKLKEAIRD-ZX-ALVEAKQARSDIEKLKEAIRD-Z X-VALVEAKQARSDIEKLKEAIRD-ZX-AVALVEAKQARSDIEKLKEAIRD-Z X-AAVALVEAKQARSDIEKLKEAIRD-ZX-TAAVALVEAKQARSDIEKLKEAIRD-Z X-ITAAVALVEAKQARSDIEKLKEAIRD-ZX-QITAAVALVEAKQARSDIEKLKEAIRD-Z X-AQITAAVALVEAKQARSDIEKLKEAIRD-ZX-SAQITAAVALVEAKQARSDIEKLKEAIRD-Z X-TSAQITAAVALVEAKQARSDIEKLKEAIRD-ZX-ATSAQITAAVALVEAKQARSDIEKLKEAIRD-Z X-VATSAQITAAVALVEAKQARSDIEKLKEAIRD-ZX-GVATSAQITAAVALVEAKQARSDIEKLKEAIRD-ZX-LGVATSAQITAAVALVEAKQARSDIEKLKEAIRD-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonylgroup; a macromolecular carrier group including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.TABLE XXIII REPRESENTATIVE DP107/DP178 ANALOG ANTIVIRAL PEPTIDESAnti-Respiratory syncytial virus peptidesX-TSVITIELSNIKENKCNGTDAKVKLIKQELDKYKN-ZX-SVITIELSNIKENKCNGTDAKVKLIKQELDKYKNA-ZX-VITIELSNIKENKCNGTDAKVKLIKQELDKYKNAV-ZX-VAVSKVLHLEGEVNKIALLSTNKAVVSLSNGVS-ZX-AVSKVLHLEGEVNKIALLSTNKAVVSLSNGVSV-ZX-VSKVLHLEGEVNKIALLSTNKAVVSLSNGVSVL-ZX-SKVLHLEGEVNKIALLSTNKAVVSLSNGVSVLT-ZX-KVLHLEGEVNKIALLSTNKAVVSLSNGVSVLTS-ZX-LEGEVNKIALLSTNKAVVSLSNGVSVLTSKVLD-ZX-GEVNKIALLSTNKAVVSLSNGVSVLTSKVLDLK-ZX-EVNKIALLSTNKAVVSLSNGVSVLTSKVLDLKN-ZX-VNKIALLSTNKAVVSLSNGVSVLTSKVLDLKNY-ZX-NKIALLSTNKAVVSLSNGVSVLTSKVLDLKNYI-ZX-KIALLSTNKAVVSLSNGVSVLTSKVLDLKNYID-ZX-IALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDK-ZX-ALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ-ZX-VAVSKVLHLEGEVNKIALLSTNKAVVSLSNGVS-ZX-AVSKVLHLEGEVNKIALLSTNKAVVSLSNGVSV-ZX-VSKVLHLEGEVNKIALLSTNKAVVSLSNGVSVL-ZX-SKVLHLEGEVNKIALLSTNKAVVSLSNGVSVLT-ZX-KVLHLEGEVNKIALLSTNKAVVSLSNGVSVLTS-ZX-LEGEVNKIALLSTNKAVVSLSNGVSVLTSKVLD-ZX-GEVNKIALLSTNKAVVSLSNGVSVLTSKVLDLK-ZX-EVNKIALLSTNKAVVSLSNGVSVLTSKVLDLKN-ZX-VNKIALLSTNKAVVSLSNGVSVLTSKVLDLKNY-ZX-NKIALLSTNKAVVSLSNGVSVLTSKVLDLKNYI-ZX-KIALLSTNKAVVSLSNGVSVLTSKVLDLKNYID-ZX-IALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDK-ZX-ALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ-Z Anti-human parainfluenza virus 3peptides X-TLNNSVALDPIDISIELNKAKSDLEESKEWIRRSN-ZX-LNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQ-ZX-NNSVALDPIDISIELNKAKSDLEESKEWIRRSNQK-ZX-NSVALDPIDISIELNKAKSDLEESKEWIRRSNQKL-ZX-SVALDPIDISIELNKAKSDLEESKEWIRRSNQKLD-ZX-VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDS-ZX-ALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI-ZX-LDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIG-ZX-DPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGN-ZX-PIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNW-ZX-IDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWH-ZX-DISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQ-ZX-ISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQS-ZX-SIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSS-ZX-IELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSST-ZX-ELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT-ZX-TAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQS-ZX-AVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSI-ZX-LVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNL-ZX-VEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLI-ZX-EAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIV-ZX-AKQARSDIEKLKEAIRDTNKAVQSVQSSICNLIVA-ZX-KQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAI-ZX-QARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIK-ZX-ARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKS-ZX-RSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSV-ZX-SDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQ-ZX-KLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVN-ZX-LKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNK-ZX-AIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIV-Z Anti-simian immunodeficiencyvirus peptides X-WQEWERKVDFLEENITALLEEAQIQQEKNMYELQK-ZX-QEWERKVDFLEENITALLEEAQIQQEKNMYELQKL-ZX-EWERKVDFLEENITALLEEAQIQQEKNMYELQKLN-ZX-WERKVDFLEENITALLEEAQIQQEKNMYELQKLNS-ZX-ERKVDFLEENITALLEEAQIQQEKNMYELQKLNSW-ZX-RKVDFLEENITALLEEAQIQQEKNMYELQKLNSWD-ZX-KVDFLEENITALLEEAQIQQEKNMYELQKLNSWDV-ZX-VDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF-ZX-DFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG-ZX-FLEENITALLEEAQIQQEKNMYELQKLNSWDVFGN-Z Anti-measles virus peptidesX-LHRIDLGPPISLERLDVGTNLCNAIAKLEAKELL-ZX-HRIDLGPPISLERLDVGTNLGNAIAKLEAKELLE-ZX-RIDLGPPISLERLDVGTNLGNAIAKLEAKELLES-ZX-IDLGPPISLERLDVGTNLGNAIAKLEAKELLESS-ZX-DLGPPISLERLDVGTNLGNAIAKLEAKELLESSD-ZX-LGPPISLERLDVGTNLGNAIAKLEAKELLESSDQ-ZX-GPPISLERLDVGTNLGNAIAKLEAKELLESSDQI-ZX-PPISLERLDVGTNLGNAIAKLEAKELLESSDQIL-ZX-PISLERLDVGTNLGNAIAKLEAKELLESSDQILR-ZX-SLERLDVGTNLGNAIAKLEAKELLESSDQILRSM-ZX-LERLDVGTNLGNAIAKLEAKELLESSDQILRSMK-Z

The one letter amino acid code is used.

Additionally,

“X” may represent an amino group, a hydrophobic group, including but notlimited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetylgroup; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates. “Z” may represent a carboxylgroup; an amido group; a T-butyloxycarbonyl group; a macromolecularcarrier group including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates.

5.4. Synthesis of Peptides

The peptides of the invention may be synthesized or prepared bytechniques well known in the art. See, for example, Creighton, 1983,Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY,which is incorporated herein by reference in its entirety. Shortpeptides, for example, can be synthesized on a solid support or insolution. Longer peptides may be made using recombinant DNA techniques.Here, the nucleotide sequences encoding the peptides of the inventionmay be synthesized, and/or cloned, and expressed according to techniqueswell known to those of ordinary skill in the art. See, for example,Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols.1-3, Cold Spring Harbor Press, NY.

The peptides of the invention may alternatively be synthesized such thatone or more of the bonds which link the amino acid residues of thepeptides are non-peptide bonds. These alternative non-peptide bonds maybe formed by utilizing reactions well known to those in the art, and mayinclude, but are not limited to imino, ester, hydrazide, semicarbazide,and azo bonds, to name but a few. In yet another embodiment of theinvention, peptides comprising the sequences described above may besynthesized with additional chemical groups present at their aminoand/or carboxy termini, such that, for example, the stability,bioavailability, and/or inhibitory activity of the peptides is enhanced.For example, hydrophobic groups such as carbobenzoxyl, dansyl, ort-butyloxycarbonyl groups, may be added to the peptides' amino termini.Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may beplaced at the peptides' amino termini. (See “X” in Tables I to IV,above.) Additionally, the hydrophobic group, t-butyloxycarbonyl, or anamido group may be added to the peptides' carboxy termini. (See “Z” inTables I to IV, above.)

Further, the peptides of the invention may be synthesized such thattheir steric configuration is altered. For example, the D-isomer of oneor more of the amino acid residues of the peptide may be used, ratherthan the usual L-isomer.

Still further, at least one of the amino acid residues of the peptidesof the invention may be substituted by one of the well knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or inhibitoryaction of the peptides of the invention.

Any of the peptides described above may, additionally, have amacromolecular carrier group covalently attached to their amino and/orcarboxy termini. Such macromolecular carrier groups may include, forexample, lipid-fatty acid conjugates, polyethylene glycol, carbohydratesor additional peptides. “X”, in Tables I to IV, above, may thereforeadditionally represent any of the above macromolecular carrier groupscovalently attached to the amino terminus of a peptide, with anadditional peptide group being preferred. Likewise, “Z”, in Tables I toIV, may additionally represent any of the macromolecular carrier groupsdescribed above.

5.5. Assays for Anti-Membrane Fusion Activity

Described herein, are methods for ability of a compound, such as thepeptides of the invention, to inhibit membrane fusion events.Specifically, assays for cell fusion events are described in Section5.5.1, below, and assays for antiviral activity are described in Section5.5.2, below.

5.5.1 Assays for Cell Fusion Events

Assays for cell fusion events are well known to those of skill in theart, and may be used in conjunction, for example, with the peptides ofthe invention to test the peptides' antifusogenic capabilities.

Cell fusion assays are generally performed in vitro. Such an assay maycomprise culturing cells which, in the absence of any treatment wouldundergo an observable level of syncytial formation. For example,uninfected cells may be incubated in the presence of cells chronicallyinfected with a virus that induces cell fusion. Such viruses mayinclude, but are not limited to, HIV, SIV, or respiratory syncytialvirus.

For the assay, cells are incubated in the presence of a peptide to beassayed. For each peptide, a range of peptide concentrations may betested. This range should include a control culture wherein no peptidehas been added.

Standard conditions for culturing cells, well known to those of ordinaryskill in the art, are used. After incubation for an appropriate period(24 hours at 37° C., for example) the culture is examinedmicroscopically for the presence of multinucleated giant cells, whichare indicative of cell fusion and syncytial formation. Well knownstains, such as crystal violet stain, may be used to facilitate thevisualization of syncytial formation.

5.5.2 Assays for Antiviral Activity

The antiviral activity exhibited by the peptides of the invention may bemeasured, for example, by easily performed in vitro assays, such asthose described below, which can test the peptides' ability to inhibitsyncytia formation, or their ability to inhibit infection by cell-freevirus. Using these assays, such parameters as the relative antiviralactivity of the peptides, exhibit against a given strain of virus and/orthe strain specific inhibitory activity of the peptide can bedetermined.

A cell fusion assay may be utilized to test the peptides' ability toinhibit viral-induced, such as HIV-induced, syncytia formation in vitro.Such an assay may comprise culturing uninfected cells in the presence ofcells chronically infected with a syncytial-inducing virus and a peptideto be assayed. For each peptide, a range of peptide concentrations maybe tested. This range should include a control culture wherein nopeptide has been added. Standard conditions for culturing, well known tothose of ordinary skill in the art, are used. After incubation for anappropriate period (24 hours at 37° C., for example) the culture isexamined microscopically for the presence of multinucleated giant cells,which are indicative of cell fusion and syncytia formation. Well knownstains, such as crystal violet stain, may be used to facilitatesyncytial visualization. Taking HIV as an example, such an assay wouldcomprise CD-4⁺ cells (such as Molt or CEM cells, for example) culturedin the presence of chronically HIV-infected cells and a peptide to beassayed.

Other well known characteristics of viral infection may also be assayedto test a peptide's antiviral capabilities. Once again taking HIV as anexample, a reverse transcriptase (RT) assay may be utilized to test thepeptides' ability to inhibit infection of CD-4⁺ cells by cell-free HIV.Such an assay may comprise culturing an appropriate concentration (i.e.,TCID₅₀) of virus and CD-4⁺ cells in the presence of the peptide to betested. Culture conditions well known to those in the art are used. Asabove, a range of peptide concentrations may be used, in addition to acontrol culture wherein no peptide has been added. After incubation foran appropriate period (e.g., 7 days) of culturing, a cell-freesupernatant is prepared, using standard procedures, and tested for thepresent of RT activity as a measure of successful infection. The RTactivity may be tested using standard techniques such as those describedby, for example, Goff et al. (Goff, S. et al., 1981, J. Virol.38:239-248) and/or Willey et al. (Willey, R. et al., 1988, J. Virol.62:139-147). These references are incorporated herein by reference intheir entirety.

Standard methods which are well-known to those of skill in the art maybe utilized for assaying non-retroviral activity. See, for example,Pringle et al. (Pringle, C. R. et al., 1985, J. Medical Virology17:377-386) for a discussion of respiratory syncytial virus andparainfluenza virus activity assay techniques. Further, see, forexample, “Zinsser Microbiology”, 1988, Joklik, W. K. et-al., eds.,Appleton & Lange, Norwalk, Conn., 19th ed., for a general review of suchtechniques. These references are incorporated by reference herein intheir entirety. In addition, the Examples presented below, in Sections17, 18, 26 and 27 each provide additional assays for the testing of acompound's antiviral capability.

In vivo assays may also be utilized to test, for example, the antiviralactivity of the peptides of the invention. To test for anti-HIVactivity, for example, the in vivo model described in Barnett et al.(Barnett, S. W. et al., 1994, Science 266:642-646) may be used.

Additionally, anti-RSV activity can be assayed in vivo via well knownmouse models. For example, RSV can be administered intranasally to miceof various inbred strains. Virus replicates in lungs of all strains, butthe highest titers are obtained in P/N, C57L/N and DBA/2N mice.Infection of BALB/c mice produces an asymptomatic bronchiolitischaracterized by lymphocytic infiltrates and pulmonary virus titers of10⁴ to 10⁵ pfu/g of lung tissue (Taylor, G. et al., 1984, Infect. Immun.43:649-655).

Cotton rat models of RSV are also well known. Virus replicates to hightiter in the nose and lungs of the cotton rat but produces few if anysigns of inflammation.

5.6. Uses of the Peptides of the Invention

The peptides of the invention may be utilized as antifusogenic orantiviral compounds, or as compounds which modulate intracellularprocesses involving coiled coil peptide structures. Further, suchpeptides may be used to identify agents which exhibit antifusogenic,antiviral or intracellular modulatory activity. Still further, thepeptides of the invention may be utilized as organism or viraltype/subtype-specific diagnostic tools.

The antifusogenic capability of the peptides of the invention mayadditionally be utilized to inhibit or treat/ameliorate symptoms causedby processes involving membrane fusion events. Such events may include,for example, virus transmission via cell-cell fusion, abnormalneurotransmitter exchange via cell-fusion, and sperm-egg fusion.Further, the peptides of the invention may be used to inhibit freeviral, such as retroviral, particularly HIV, transmission to uninfectedcells wherein such viral infection involves membrane fusion events orinvolves fusion of a viral structure with a cell membrane. Among theintracellular disorders involving coiled coil peptides structures whichmay be ameliorated by the peptides of the invention are disordersinvolving, for example, bacterial toxins.

With respect to antiviral activity, the viruses whose transmission maybe inhibited by the peptides of the invention include, but are notlimited to all strains of the viruses listed above, in Tables V throughVII, and 1× through XIV.

These viruses include, for example, human retroviruses, particularlyHIV-1 and HIV-2 and the human T-lymphocyte viruses (HTLV-I and II). Thenon-human retroviruses whose transmission may be inhibited by thepeptides of the invention include, but are not limited to bovineleukosis virus, feline sarcoma and leukemia viruses, simianimmunodeficiency, sarcoma and leukemia viruses, and sheep progresspneumonia viruses.

Non retroviral viruses whose transmission may be inhibited by thepeptides of the invention include, but are not limited to humanrespiratory syncytial virus, canine distemper virus, newcastle diseasevirus, human parainfluenza virus., influenza viruses, measles viruses,Epstein-Barr viruses, hepatitis B viruses, and simian Mason-Pfizerviruses.

Non enveloped viruses whose transmission may be inhibited by thepeptides of the invention include, but are not limited to picornavirusessuch as polio viruses, hepatitis A virus, enterovirus, echoviruses andcoxsackie viruses, papovaviruses such as papilloma virus, parvoviruses,adenoviruses and reoviruses.

As discussed more fully, below, in Section 5.5.1 and in the Examplepresented, below, in Section 8, DP107, DP178, DP107 analog and DP178analog peptides form non-covalent protein-protein interactions which arerequired for normal activity of the virus. Thus, the peptides of theinvention may also be utilized as components in assays for theidentification of compounds that interfere with such protein-proteininteractions and may, therefore, act as antiviral agents. These assaysare discussed, below, in Section 5.5.1.

As demonstrated in the Example presented below in Section 6, theantiviral activity of the peptides of the invention may show apronounced type and subtype specificity, i.e., specific peptides may beeffective in inhibiting the activity of only specific viruses. Thisfeature of the invention presents many advantages. One such advantage,for example, lies in the field of diagnostics, wherein one can use theantiviral specificity of the peptide of the invention to ascertain theidentity of a viral isolate. With respect to HIV, one may easilydetermine whether a viral isolate consists of an HIV-1 or HIV-2 strain.For example, uninfected CD-4⁺ cells may be co-infected with an isolatewhich has been identified as containing HIV the DP178 (SEQ ID:1)peptide, after which the retroviral activity of cell supernatants may beassayed, using, for example, the techniques described above in Section5.2. Those isolates whose retroviral activity is completely or nearlycompletely inhibited contain HIV-1. Those isolates whose viral activityis unchanged or only reduced by a small amount, may be considered to notcontain HIV-1. Such an isolate may then be treated with one or more ofthe other DP178 peptides of the invention, and subsequently be testedfor its viral activity in order to determine the identify of the viralisolate. The DP107 and DP178 analogs of the invention may also beutilized in a diagnostic capacity specific to the type and subtype ofvirus or organism in which the specific peptide sequence is found. Adiagnostic procedure as described, above, for DP178, may be used inconjunction with the DP107/DP178 analog of interest.

5.5.1. Screening Assays

As demonstrated in the Example presented in Section 8, below, DP107 andDP178 portions of the TM protein gp41 form non-covalent protein-proteininteractions. As is also demonstrated, the maintenance of suchinteractions is necessary for normal viral infectivity. Thus, compoundswhich bind DP107, bind DP178, and/or act to disrupt normal DP107/DP178protein-protein interactions may act as antifusogenic, antiviral orcellular modulatory agents. Described below are assays for theidentification of such compounds. Note that, while, for ease and clarityof discussion, DP107 and DP178 peptides will be used as components ofthe assays described, but it is to be understood that any of the DP107analog or DP178 analog peptides described, above, in Sections 5.1through 5.3 may also be utilized as part of these screens for compounds.

Compounds which may be tested for an ability to bind DP107, DP178,and/or disrupt DP107/DP178 interactions, and which therefore,potentially represent antifusogenic, antiviral or intracellularmodulatory compounds, include, but are not limited to, peptides made ofD- and/or L-configuration amino acids (in, for example, the form ofrandom peptide libraries; see Lam, K. S. et al., 1991, Nature354:82-84), phosphopeptides (in, for example, the form of random orpartially degenerate, directed phosphopeptide libraries; see, forexample, Songyang, Z. et al., 1993, Cell 72:767-778), antibodies, andsmall organic or inorganic molecules. Synthetic compounds, naturalproducts, and other sources of potentially effective materials may bescreened in a variety of ways, as described in this Section.

The compounds, antibodies, or other molecules identified may be tested,for example, for an ability to inhibit cell fusion or viral activity,utilizing, for example, assays such as those described, above, inSection 5.5.

Among the peptides which may be tested are soluble peptides comprisingDP107 and/or DP178 domains, and peptides comprising DP107 and/or DP178domains having one or more mutations within one or both of the domains,such as the M41-P peptide described, below, in the Example presented inSection 8, which contains a isoleucine to proline mutation within theDP178 sequence.

In one embodiment of such screening methods is a method for identifyinga compound to be tested for antiviral ability comprising:

-   -   (a) exposing at least one compound to a peptide comprising a        DP107 peptide for a time sufficient to allow binding of the        compound to the DP107 peptide;    -   (b) removing non-bound compounds; and    -   (c) determining the presence of the compound bound to the DP107        peptide, thereby identifying an agent to be tested for antiviral        ability.

In a second embodiment of such screening methods is a method foridentifying a compound to be tested for antiviral ability comprising:

-   -   (a) exposing at least one compound to a peptide comprising a        DP178 peptide for a time sufficient to allow binding of the        compound to the DP178 peptide;    -   (b) removing non-bound compounds; and    -   (c) determining the presence of the compound bound to the DP178        peptide, thereby identifying an agent to be tested for antiviral        ability.

One method utilizing these types of approaches that may be pursued inthe isolation of such DP107-binding or DP178-binding compounds is anassay which would include the attachment of either the DP107 or theDP178 peptide to a solid matrix, such as, for example, agarose orplastic beads, microtiter plate wells, petri dishes, or membranescomposed of, for example, nylon or nitrocellulose. In such an assaysystem, either the DP107 or DP178 protein may be anchored onto a solidsurface, and the compound, or test substance, which is not anchored, islabeled, either directly or indirectly. In practice, microtiter platesare conveniently utilized. The anchored component may be immobilized bynon-covalent or covalent attachments. Non-covalent attachment may beaccomplished simply by coating the solid surface with a solution of theprotein and drying.

Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein may be used to anchor the protein tothe solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the labeled compound is added to thecoated surface containing the anchored DP107 or DP178 peptide. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thecompound is pre-labeled, the detection of label immobilized on thesurface indicates that complexes were formed. Where the labeledcomponent is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the compound (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, such an assay can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for DP107 orDP178, whichever is appropriate for the given assay, or ab antibodyspecific for the compound, i.e., the test substance, in order to anchorany complexes formed in solution, and a labeled antibody specific forthe other member of the complex to detect anchored complexes.

By utilizing procedures such as this, large numbers of types ofmolecules may be simultaneously screened for DP107 or DP178-bindingcapability, and thus potential antiviral activity.

Further, compounds may be screened for an ability to inhibit theformation of or, alternatively, disrupt DP107/DP178 complexes. Suchcompounds may then be tested for antifusogenic, antiviral orintercellular modulatory capability. For ease of description, DP107 andDP178 will be referred to as “binding partners.” Compounds that disruptsuch interactions may exhibit antiviral activity. Such compounds mayinclude, but are not limited to molecules such as antibodies, peptides,and the like described above.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the DP107 and DP178 peptidesinvolves preparing a reaction mixture containing peptides underconditions and for a time sufficient to allow the two peptides tointeract and bind, thus forming a complex. In order to test a compoundfor disruptive activity, the reaction is conducted in the presence andabsence of the test compound, i.e., the test compound may be initiallyincluded in the reaction mixture, or added at a time subsequent to theaddition of one of the binding partners; controls are incubated withoutthe test compound or with a placebo. The formation of any complexesbetween the binding partners is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound indicates that the compound interferes withthe interaction of the DP107 and DP178 peptides.

The assay for compounds that interfere with the interaction of thebinding partners can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring one of the bindingpartners onto a solid phase and detecting complexes anchored on thesolid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with thebinding partners. On the other hand, test compounds that disruptpreformed complexes, e.g. compounds with higher binding constants thatdisplace one of the binding partners from the complex, can be tested byadding the test compound to the reaction mixture after complexes havebeen formed. The various formats are described briefly below.

In a heterogeneous assay system, one binding partner, e.g., either theDP107 or DP178 peptide, is anchored onto a solid surface, and itsbinding partner, which is not anchored, is labeled, either directly orindirectly. In practice, microtiter plates are conveniently utilized.The anchored species may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished simply bycoating the solid surface with a solution of the protein and drying.Alternatively, an immobilized antibody specific for the protein may beused to anchor the protein to the solid surface. The surfaces may beprepared in advance and stored.

In order to conduct the assay, the binding partner of the immobilizedspecies is added to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thebinding partner was pre-labeled, the detection of label immobilized onthe surface indicates that complexes were formed. Where the bindingpartner is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the binding partner (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one binding partner to anchor anycomplexes formed in solution, and a labeled antibody specific for theother binding partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the DP107 and DP178peptides is prepared in which one of the binding partners is labeled,but the signal generated by the label is quenched due to complexformation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein whichutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the binding partnersfrom the preformed complex will result in the generation of a signalabove background. In this way, test substances which disruptDP-107/DP-178 protein-protein interaction can be identified.

In an alternative screening assay, test compounds may be assayed for thetheir ability to disrupt a DP178/DP107 interaction, as measuredimmunometrically using an antibody specifically reactive to aDP107/DP178 complex (i.e., an antibody that recognizes neither DP107 norDP178 individually). Such an assay acts as a competition assay, and isbased on techniques well known to those of skill in the art.

The above competition assay may be described, by way of example, and notby way of limitation, by using the DP178 and M41A178 peptides and byassaying test compounds for the disruption of the complexes formed bythese two peptides by immunometrically visualizing DP178/M41Δ178complexes via the human recombinant Fab, Fab-d, as described, below, inthe Example presented in Section 8. M41Δ178 is a maltose binding fusionprotein containing a gp41 region having its DP178 domain deleted, and isdescribed, below, in the Example presented in Section 8.

Utilizing such an assay, M41Δ178 may be immobilized onto solid supportssuch as microtiter wells. A series of dilutions of a test compound maythen be added to each M41Δ178-containing well in the presence of aconstant concentration of DP-178 peptide. After incubation, at, forexample, room temperature for one hour, unbound DP-178 and test compoundare removed from the wells and wells are then incubated with theDP178/M41Δ178-specific Fab-d antibody. After incubation and washing,unbound Fab-d is removed from the plates and bound Fab-d is quantitated.A no-inhibitor control should also be conducted. Test compounds showingan ability to disrupt DP178/M41Δ178 complex formation are identified bytheir concentration-dependent decrease in the level of Fab-d binding.

A variation of such an assay may be utilized to perform a rapid,high-throughput binding assay which is capable of directly measuringDP178 binding to M41Δ178 for the determination of binding constants ofthe ligand of inhibitory constants for competitors of DP178 binding.

Such an assay takes advantage of accepted radioligand and receptorbinding principles. (See, for example, Yamamura, H. I. et al., 1985,“Neurotransmitter Receptor Binding”, 2nd ed., Raven Press, NY.) Asabove, M41Δ178 is immobilized onto a solid support such as a microtiterwell. DP178 binding to M41Δ178 is then quantitated by measuring thefraction of DP178 that is bound as ¹²⁵I]-DP178 and calculating the totalamount bound using a value for specific activity (dpm/μg peptide)determined for each labeled DP178 preparation. Specific binding toM41Δ178 is defined as the difference of the binding of the labeled DP178preparation in the microtiter wells (totals) and the binding inidentical wells containing, in addition, excess unlabeled DP178(nonspecifics).

5.5 Pharmaceutical Formulations, Dosages and Modes of Administration

The peptides of the invention may be administered using techniques wellknown to those in the art. Preferably, agents are formulated andadministered systemically. Techniques for formulation and administrationmay be found in “Remington's Pharmaceutical Sciences”, 18th ed., 1990,Mack Publishing Co., Easton, Pa. Suitable routes may include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as, intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew. For injection, the agents of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.For such transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

In instances wherein intracellular administration of the peptides of theinvention or other inhibitory agents is preferred, techniques well knownto those of ordinary skill in the art may be utilized. For example, suchagents may be encapsulated into liposomes, then administered asdescribed above. Liposomes are spherical lipid bilayers with aqueousinteriors. All molecules present in an aqueous solution at the time ofliposome formation are incorporated into the aqueous interior. Theliposomal contents are both protected from the external microenvironmentand, because liposomes fuse with cell membranes, are effectivelydelivered into the cell cytoplasm. Additionally, due to theirhydrophobicity, when small molecules are to be administered, directintracellular administration may be achieved.

Nucleotide sequences encoding the peptides of the invention which are tobe intracellularly administered may be expressed in cells of interest,using techniques well known to those of skill in the art. For example,expression vectors derived from viruses such as retroviruses, vacciniaviruses, adeno-associated viruses, herpes viruses, or bovine papillomaviruses, may be used for delivery and expression of such nucleotidesequences into the targeted cell population. Methods for theconstruction of such vectors and expression constructs are well known.See, for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor N.Y., and Ausubelet al., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, NY.

With respect to HIV, peptides of the invention, particularly DP107 andDP178, may be used as therapeutics in the treatment of AIDS. Inaddition, the peptides may be used as prophylactic measures inpreviously uninfected individuals after acute exposure to an HIV virus.Examples of such prophylactic use of the peptides may include, but arenot limited to, prevention of virus transmission from mother to infantand other settings where the likelihood of HIV transmission exists, suchas, for example, accidents in health care settings wherein workers areexposed to HIV-containing blood products. The successful use of suchtreatments do not rely upon the generation of a host immune responsedirected against such peptides.

Effective dosages of the peptides of the invention to be administeredmay be determined through procedures well known to those in the artwhich address such parameters as biological half-life, bioavailability,and toxicity. Given the data presented below in Section 6, DP178, forexample, may prove efficacious in vivo at doses required to achievecirculating levels of about 1 to about 10 ng per ml of peptide.

A therapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in humans. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (e.g., theconcentration of the test compound which achieves a half-maximalinhibition of the fusogenic event, such as a half-maximal inhibition ofviral infection relative to the amount of the event in the absence ofthe test compound) as determined in cell culture. Such information canbe used to more accurately determine useful doses in humans. Levels inplasma may be measured, for example, by high performance liquidchromatography (HPLC).

The peptides of the invention may, further, serve the role of aprophylactic vaccine, wherein the host raises antibodies against thepeptides of the invention, which then serve to neutralize HIV virusesby, for example, inhibiting further HIV infection.

Administration of the peptides of the invention as a prophylacticvaccine, therefore, would comprise administering to a host aconcentration of peptides effective in raising an immune response whichis sufficient to neutralize HIV, by, for example, inhibiting HIV abilityto infect cells. The exact concentration will depend upon the specificpeptide to be administered, but may be determined by using standardtechniques for assaying the development of an immune response which arewell known to those of ordinary skill in the art. The peptides to beused as vaccines are usually administered intramuscularly.

The peptides may be formulated with a suitable adjuvant in order toenhance the immunological response. Such adjuvants may include, but arenot limited to mineral gels such as aluminum hydroxide; surface activesubstances such as lysolecithin, pluronic polyols, polyanions; otherpeptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum. Many methods may be used to introducethe vaccine formulations described here. These methods include but arenot limited to oral, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, and intranasal routes.

Alternatively, an effective concentration of polyclonal or monoclonalantibodies raised against the peptides of the invention may beadministered to a host so that no uninfected cells become infected byHIV. The exact concentration of such antibodies will vary according toeach specific antibody preparation, but may be determined using standardtechniques well known to those of ordinary skill in the art.Administration of the antibodies may be accomplished using a variety oftechniques, including, but not limited to those described in thissection.

For all such treatments described above, the exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g. Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the oncogenic disorder of interest will varywith the severity of the condition to be treated and the route ofadministration. The dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

6. EXAMPLE DP178 (SEQ ID:1) is a Potent Inhibitor of HIV-1 Infection

In this example, DP178 (SEQ ID:1) is shown to be a potent inhibitor ofHIV-1 mediated CD-4⁺ cell-cell fusion and infection by cell free virus.In the fusion assay, this peptide completely blocks virus inducedsyncytia formation at concentrations of from 1-10 ng/ml. In theinfectivity assay the inhibitory concentration is somewhat higher,blocking infection at 90 ng/ml. It is further shown that DP178 (SEQID:1) shows that the antiviral activity of DP178 (SEQ ID:1) is highlyspecific for HIV-1. Additionally, a synthetic peptide, DP-185 (SEQID:3), representing a HIV-1-derived DP178 homolog is also found to blockHIV-1-mediated syncytia formation.

6.1. Materials and Methods 6.1.1. Peptide Synthesis

Peptides were synthesized using Fast Moc chemistry on an AppliedBiosystems Model 431A peptide synthesizer. Generally, unless otherwisenoted, the peptides contained amidated carboxy termini and acetylatedamino termini. Amidated peptides were prepared using Rink resin(Advanced Chemtech) while peptides containing free carboxy termini weresynthesized on Wang (p-alkoxy-benzyl-alcohol) resin (Bachem). Firstresidues were double coupled to the appropriate resin and subsequentresidues were single coupled. Each coupling step was followed by aceticanhydride capping. Peptides were cleaved from the resin by treatmentwith trifluoracetic acid (TFA) (10 ml), H₂O (0.5 ml), thioanisole (0.5ml), ethanedithiol (0.25 ml), and crystalline phenol (0.75 g).Purification was carried out by reverse phase HPLC. Approximately 50 mgsamples of crude peptide were chromatographed on a Waters Delta Pak C18column (19 mm×30 cm, 15μ spherical) with a linear gradient;H₂O/acetonitrile 0.1% TFA. Lyophilized peptides were stored desiccatedand peptide solutions were made in water at about 1 mg/ml. Electrospraymass spectrometry yielded the following results: DP178 (SEQID:1):4491.87 (calculated 4491.94); DP-180 (SEQ ID:2):4491.45(calculated 4491.94); DP-185 (SEQ ID:3):not done (calculated 4546.97).

6.1.2. Virus

The HIV-1_(LAI) virus was obtained from R. Gallo (Popovic, M. et al.,1984, Science 224:497-508) and propagated in CEM cells cultured in RPMI1640 containing 10% fetal calf serum. Supernatant from the infected CEMcells was passed through a 0.2 μm filter and the infectious titerestimated in a microinfectivity assay using the AA5 cell line to supportvirus replication. For this purpose, ²⁵ μl of serial diluted virus wasadded to 75 μl AA5 cells at a concentration of 2×10⁵/ml in a 96-wellmicrotitre plate. Each virus dilution was tested in triplicate. Cellswere cultured for eight days by addition of fresh medium every otherday. On day 8 post infection, supernatant samples were tested for virusreplication as evidenced by reverse transcriptase activity released tothe supernatant. The TCID₅₀ was calculated according to the Reed andMuench formula (Reed, L. J. et al., 1938, Am. J. Hyg. 27:493-497). Thetiter of the HIV-1_(LAI) and HIV-1_(MN) stocks used for these studies,as measured on the AA5 cell line, was approximately 1.4×10⁶ and 3.8×10⁴TCID₅₀/ml, respectively.

6.1.3. Cell Fusion Assay

Approximately 7×10⁴ Molt cells were incubated with 1×10⁴ CEM cellschronically infected with the HIV-1_(LAI) virus in 96-well plates(one-half area cluster plates; Costar, Cambridge, Mass.) in a finalvolume of 100 μl culture medium as previously described (Matthews, T. J.et al., 1987, Proc. Natl. Acad. Sci. USA 84: 5424-5428). Peptideinhibitors were added in a volume of 10 μl and the cell mixtures wereincubated for 24 hr. at 37° C. At that time, multinucleated giant cellswere estimated by microscopic examination at a 40× magnification whichallowed visualization of the entire well in a single field.

6.1.4. Cell Free Virus Infection Assay

Synthetic peptides were incubated at 37° C. with either 247 TCID₅₀ (forexperiment depicted in FIG. 2), or 62 TCID₅₀ (for experiment depicted inFIG. 3) units of HIV-1_(LAI) virus or 25 TCID₅₀ units of HIV-2_(NIHZ)and CEM CD4⁺ cells at peptide concentrations of 0, 0.04, 0.4, 4.0, and40 μg/ml for 7 days. The resulting reverse transcriptase (RT) activityin counts per minute was determined using the assay described, below, inSection 6.1.5. See, Reed, L. J. et al., 1938, Am. J. Hyg. 27: 493-497for an explanation of TCID₅₀ calculations.

6.1.5. Reverse Transcriptase Assay

The micro-reverse transcriptase (RT) assay was adapted from Goff et al.(Goff, S. et al., 1981, J. Virol. 38:239-248) and Willey et al. (Willey,R. et al., 1988, J. Virol. 62:139-147). Supernatants from virus/cellcultures are adjusted to 1% Triton-X100. A 10 μl sample of supernatantwas added to 50 μl of RT cocktail in a 96-well U-bottom microtitre plateand the samples incubated at 37° C. for 90 min. The RT cocktailcontained 75 mM KCl, 2 mM dithiothreitol, 5 mM MgCl₂, 5 μg/ml poly A(Pharmacia, cat. No. 27-4110-01), 0.25 units/ml oligo dT (Pharmacia,cat. No. 27-7858-01), 0.05% NP40, 50 mM Tris-HCl, pH 7.8, 0.5 μMnon-radioactive dTTP, and 10 μCi/ml ³²P-dTTP (Amersham, cat. No. PB.10167).

After the incubation period, 40 μl of reaction mixture was applied to aSchleicher and Schuell (S+S) NA45 membrane (or DE81 paper) saturated in2×SSC buffer (0.3M NaCl and 0.003M sodium citrate) held in a S+SMinifold over one sheet of GB003 (S+S) filter paper, with partial vacuumapplied. Each well of the minifold was washed four times with 200 μl2×SSC, under full vacuum. The membrane was removed from the minifold andwashed 2 more times in a pyrex dish with an excess of 2×SSC. Finally,the membrane was drained on absorbent paper, placed on Whatman #3 paper,covered with Saran wrap, and exposed to film overnight at −70° C.

6.2. Results 6.2.1. Peptide Inhibition of Infected Cell-Induced SyncytiaFormation

The initial screen for antiviral activity assayed peptides' ability toblock syncytium formation induced by overnight co-cultivation ofuninfected Molt4 cells with chronically HIV-1 infected CEM cells. Theresults of several such experiments are presented herein. In the firstof these experiments, serial DP178 (SEQ ID:1) peptide concentrationsbetween 10 μg/ml and 12.5 ng/ml were tested for blockade of the cellfusion process. For these experiments, CEM cells chronically infectedwith either HIV-1_(LAI), HIV-1_(MN), HIV-1_(RF), or HIV-1_(SF2) viruswere cocultivated overnight with uninfected Molt 4 cells. The results(FIG. 4) show that DP178 (SEQ ID:1) afforded complete protection againsteach of the HIV-1 isolates down to the lowest concentration of DP178(SEQ ID:1) used. For HIV_(LAI) inhibition, the lowest concentrationtested was 12.5 ng/ml; for all other HIV-1 viruses, the lowestconcentration of DP178 (SEQ ID:1) used in this study was 100 ng/ml. Asecond peptide, DP-180 (SEQ ID:2), containing the same amino acidresidues as DP178 (SEQ ID:1) but arranged in a random order exhibited noevidence of anti-fusogenic activity even at the high concentration of 40μg/ml (FIG. 4). These observations indicate that the inhibitory effectof DP178 (SEQ ID:1) is primary sequence-specific and not related tonon-specific peptide/protein interactions. The actual endpoint (i.e.,the lowest effective inhibitory concentration) of DP178 inhibitoryaction is within the range of 1-10 ng/ml.

The next series of experiments involved the preparation and testing of aDP178 (SEQ ID:1) homolog for its ability to inhibit HIV-1-inducedsyncytia formation. As shown in FIG. 1, the sequence of DP-185 (SEQID:3) is slightly different from DP178 (SEQ ID:1) in that its primarysequence is taken from the HIV-1_(SF2) isolate and contains severalamino acid differences relative to DP178 (SEQ ID:1) near the N terminus.As shown in FIG. 4, DP-185 (SEQ ID:3), exhibits inhibitory activity evenat 312.5 ng/ml, the lowest concentration tested.

The next series of experiments involved a comparison of DP178 (SEQ ID:1)HIV-1 and HIV-2 inhibitory activity. As shown in FIG. 5, DP178 (SEQID:1) blocked HIV-1-mediated syncytia formation at peptideconcentrations below 1 ng/ml. DP178 (SEQ ID:1) failed, however, to blockHIV-2 mediated syncytia formation at concentrations as high as 10 μg/ml.This striking 4 log selectivity of DP178 (SEQ ID:1) as an inhibitor ofHIV-1-mediated cell fusion demonstrates an unexpected HIV-1 specificityin the action of DP178 (SEQ ID:1). DP178 (SEQ ID:1) inhibition ofHIV-1-mediated cell fusion, but the peptide's inability to inhibit HIV-2medicated cell fusion in the same cell type at the concentrations testedprovides further evidence for the high degree of selectivity associatedwith the antiviral action of DP178 (SEQ ID:1).

6.2.2. Peptide Inhibition of Infection by Cell-Free Virus

DP178 (SEQ ID:1) was next tested for its ability to block CD-4⁺ CEM cellinfection by cell free HIV-1 virus. The results, shown in FIG. 2, arefrom an experiment in which DP178 (SEQ ID:1) was assayed for its abilityto block infection of CEM cells by an HIV-1_(LAI) isolate. Included inthe experiment were three control peptides, DP-116 (SEQ ID:9), DP-125(SEQ ID:8), and DP-118 (SEQ ID:10). DP-116 (SEQ ID:9) represents apeptide previously shown to be inactive using this assay, and DP-125(SEQ ID:8; Wild, C. et al., 1992, Proc. Natl. Acad, Sci. USA 89:10,537)and DP-118 (SEQ ID:10) are peptides which have previously been shown tobe active in this assay. Each concentration (0, 0.04, 0.4, 4, and 40g/ml) of peptide was incubated with 247 TCID₅₀ units of HIV-1_(LAI)virus and CEM cells. After 7 days of culture, cell-free supernatant wastested for the presence of RT activity as a measure of successfulinfection. The results, shown in FIG. 2, demonstrate that DP178 (SEQID:1) inhibited the de novo infection process mediated by the HIV-1viral isolate at concentrations as low as 90 ng/ml (IC50=90 ng/ml). Incontrast, the two positive control peptides, DP-125 (SEQ: ID:8) andDP-118 (SEQ ID:10), had over 60-fold higher IC50 concentrations ofapproximately 5 μg/ml.

In a separate experiment, the HIV-1 and HIV-2 inhibitory action of DP178(SEQ ID:1) was tested with CEM cells and either HIV-1_(LAI) orHIV-2_(NIHz). 62 TCID₅₀ HIV-1_(LAI) or 25 GCID₅₀ HIV-2_(NIHz) were usedin these experiments, and were incubated for 7 days. As may be seen inFIG. 3, DP178 (SEQ ID:1) inhibited HIV-1 infection with an IC50 of about31 ng/ml. In contrast, DP178 (SEQ ID:1) exhibited a much higher IC50 forHIV-2_(NIHZ), thus making DP178 (SEQ ID:1) two logs more potent as aHIV-1 inhibitor than a HIV-2 inhibitor. This finding is consistent withthe results of the fusion inhibition assays described, above, in Section6.2.1, and further supports a significant level of selectivity (i.e.,for HIV-1 over HIV-2).

7. EXAMPLE The HIV-1 Inhibitor, DP178 (SEQ ID:1) is Non-Cytotoxic

In this Example, the 36 amino acid synthetic peptide inhibitor DP178(SEQ ID:1) is shown to be non-cytotoxic to cells in culture, even at thehighest peptide concentrations (40 μg/ml) tested.

7.1. Materials and Methods

Cell proliferation and toxicity assay: Approximately 3.8×10⁵ CEM cellsfor each peptide concentration were incubated for 3 days at 37° C. inT25 flasks. Peptides tested were DP178 (SEQ ID:1) and DP-116 (SEQ ID:9),as described in FIG. 1. Peptides were synthesized as described, above,in Section 6.1. The concentrations of each peptide used were 0, 2.5, 10,and 40 g/ml. Cell counts were taken at incubation times of 0, 24, 48,and 72 hours.

7.2. Results

Whether the potent HIV-1 inhibitor DP178 (SEQ ID:1) exhibited anycytotoxic effects was assessed by assaying the peptide's effects on theproliferation and viability of cells in culture. CEM cells wereincubated in the presence of varying concentrations of DP178 (SEQ ID:1),and DP-116 (SEQ ID:9), a peptide previously shown to be ineffective as aHIV inhibitor (Wild, C. et al., 1992, Proc. Natl. Acad. Sci. USA89:10,541). Additionally, cells were incubated in the absence of eitherpeptide.

The results of the cytotoxicity study demonstrate that DP178 (SEQ ID:1)exhibits no cytotoxic effects on cells in culture. As can be seen,below, in Table XXIV, even the proliferation and viabilitycharacteristics of cells cultured for 3 days in the presence of thehighest concentration of DP178 (SEQ ID:1) tested (40 μg/ml) do notsignificantly differ from the DP-116 (SEQ ID:9) or the no peptidecontrols. The cell proliferation data is also represented in graphicform in FIG. 6. As was demonstrated in the Working Example presentedabove in Section 6, DP178 (SEQ ID:1) completely inhibits HIV-1 mediatedsyncytia formation at peptide concentrations between 1 and 10 ng/ml, andcompletely inhibits cell-free viral infection at concentrations of atleast 90 ng/ml. Thus, this study demonstrates that even at peptideconcentrations greater than 3 log higher than the HIV inhibitory dose,DP178 (SEQ ID:1) exhibits no cytotoxic effects. TABLE XXIV % ViabilityPeptide at time (hours) Peptide Concentration μg/ml 0 24 48 72 DP178 4098 97 95 97 (SEQ ID: 1) 10 98 97 98 98 2.5 98 93 96 96 DP116 40 98 95 9897 (SEQ ID: 9) 10 98 95 93 98 2.5 98 96 98 99 No 0 98 97 99 98 Peptide

8. EXAMPLE The Interaction of DP178 and DP107

Soluble recombinant forms of gp41 used in the example described belowprovide evidence that the DP178 peptide associates with a distal site ongp41 whose interactive structure is influenced by the DP107 leucinezipper motif. A single mutation disrupting the coiled-coil structure ofthe leucine zipper domain transformed the soluble recombinant gp41protein from an inactive to an active inhibitor of HIV-1 fusion. Thistransformation may result from liberation of the potent DP178 domainfrom a molecular clasp with the leucine zipper, DP107, determinant. Theresults also indicate that the anti-HIV activity of various gp41derivatives (peptides and recombinant proteins) may be due to theirability to form complexes with viral gp41 and interfere with itsfusogenic process.

8.1. Materials and Methods 8.1.1. Construction of Fusion Proteins andGP41 Mutants

Construction of fusion proteins and mutants shown in FIG. 7 wasaccomplished as follows: the DNA sequence corresponding to theextracellular domain of gp41 (540-686) was cloned into the Xmn I site ofthe expression vector pMal-p2 (New England Biolab) to give M41. The gp41sequence was amplified from pgtat (Malim et al., 1988, Nature 355:181-183) by using polymerase chain reaction (PCR) with upstream primer5′-ATGACGCTGACGGTACAGGCC-3′ (primer A) and downstream primer5′-TGACTAAGCTTAATACCACAGCCAATTTGTTAT-3′ (primer B). M41-P wasconstructed by using the T7-Gen in vitro mutagenesis kit from UnitedStates Biochemicals (USB) following the supplier's instructions. Themutagenic primer (5′-GGAGCTGCTTGGGGCCCCAGAC-3′) introduces an Ile to Promutation in M41 at position 578. M41Δ107, from which the DP-107 regionhas been deleted, was made using a deletion mutagenic primer5′-CCAAATCCCCAGGAGCTGCTCGAGCTGCACTATACCAGAC-3′ (primer C) following theUSB T7-Gen mutagenesis protocol. M41Δ178, from which the DP-178 regionhas been deleted, was made by cloning the DNA fragment corresponding togp41 amino acids 540-642 into the Xmn I site of pMal-p2. Primer A and5′-ATAGCTTCTAGATTAATTGTTAATTTCTCTGTCCC-3′ (primer D) were used in thePCR with the template pgtat to generate the inserted DNA fragments.M41-P was used as the template with primer A and D in PCR to generateM41-PΔ178. All inserted sequences and mutated residues were checked byrestriction enzyme analysis and confirmed by DNA sequencing.

8.1.2. Purification and Characterization of Fusion Proteins

The fusion proteins were purified according to the protocol described inthe manufacturer's brochure of protein fusion and purification systemsfrom New England Biolabs (NEB). Fusion proteins (10 ng) were analyzed byelectrophoresis on 8% SDS polyacrylamide gels. Western blotting analysiswas performed as described by Sambrook et al., 1989, Molecular Cloning:A Laboratory Manual, 2d Ed, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., Ch. 18, pp. 64-75. An HIV-1 positive serum diluted1000-fold, or a human Fab derived from repertoire cloning was used toreact with the fusion proteins. The second antibody was HRP-conjugatedgoat antihuman Fab. An ECL Western blotting detection system (Amersham)was used to detect the bound antibody. A detailed protocol for thisdetection system was provided by the manufacturer. Rainbow molecularweight markers (Amersham) were used to estimate the size of fusionproteins.

8.1.3. Cell Fusion Assays for Anti-HIV Activity

Cell fusion assays were performed as previously described (Matthews etal., 1987, Proc. Natl. Acad. Sci. USA 84: 5424-5481). CEM cells (7×10⁴)were incubated with HIV-1_(IIIB) chronically infected CEM cells (10⁴) in96-well flat-bottomed half-area plates (Costar) in 100 μl culturemedium. Peptide and fusion proteins at various concentrations in 10 μlculture medium were incubated with the cell mixtures at 37° C. for 24hours. Multinucleated syncytia were estimated with microscopicexamination. Both M41 and M41-P did not show cytotoxicity at theconcentrations tested and shown in FIG. 8.

Inhibition of HIV-1 induced cell-cell fusion activity was carried out inthe presence of 10 nM DP178 and various concentrations of M41Δ178 orM41-PΔ178 as indicated in FIG. 9. There was no observable syncytia inthe presence of 10 nM DP178. No peptide or fusion protein was added inthe control samples.

8.1.4. Elisa Analysis of DP178 Binding to the Leucine Zipper Motif ofGP41

The amino acid sequence of DP178 used is:YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF. For enzyme linked immunoassay(ELISA), M41Δ178 or M41-PΔ178 (5 μg/ml) in 0.1M NaHCO₃, pH 8.6, werecoated on 96 wells Linbro ELISA plates (Flow Lab, Inc.) overnight. Eachwell was washed three times with distilled water then blocked with 3%bovine serum albumin (BSA) for 2 hours. After blocking, peptides with0.5% BSA in TBST (40 mM Tris-HCl pH7.5, 150 mM NaCl, 0.05% Tween 20)were added to the ELISA plates and incubated at room temperature for 1hour. After washing three times with TBST, Fab-d was added at aconcentration of 10 ng/ml with 0.5% BSA in TBST. The plates were washedthree times with TBST after incubation at room temperature for 1 hour.Horse radish peroxidase (HRP) conjugated goat antihuman Fab antiserum ata 2000 fold dilution in TBST with 0.5% BSA was added to each well andincubated at room temperature for 45 minutes. The plates were thenwashed four times with TBST. The peroxidase substrate o-phenylenediamine (2.5 mg/ml) and 0.15% H₂O₂ were added to develop the color. Thereaction was stopped with an equal volume of 4.5 N H₂SO₄ afterincubation at room temperature for 10 minutes. The optical density ofthe stopped reaction mixture was measured with a micro plate reader(Molecular Design) at 490 nm. Results are shown in FIG. 10.

8.2. Results 8.2.1. The Expression and Characterization of TheEctodomain of gp41

As a step toward understanding the roles of the two helical regions ingp41 structure and function, the ectodomain of gp41 was expressed as amaltose binding fusion protein (M41) (FIG. 7). The fusogenic peptidesequence at the N-terminal of gp41 was omitted from this recombinantprotein and its derivatives to improve solubility. The maltose bindingprotein facilitated purification of the fusion proteins under relativelymild, non-denaturing conditions. Because the M41 soluble recombinantgp41 was not glycosylated, lacked several regions of the transmembraneprotein (i.e., the fusion peptide, the membrane spanning, and thecytoplasmic domains), and was expressed in the absence of gp120, it wasnot expected to precisely reflect the structure of native gp41 on HIV-1virions. Nevertheless, purified M41 folded in a manner that preservedcertain discontinuous epitopes as evidenced by reactivity with humanmonoclonal antibodies, 98-6, 126-6, and 50-69, previously shown to bindconformational epitopes on native gp41 expressed in eukaryotic cells (Xuet al., 1991, J. Virol. 65: 4832-4838; Chen, 1994, J. Virol.68:2002-2010). Thus, at least certain regions of native gp41 defined bythese antibodies appear to be reproduced in the recombinant fusionprotein M41. Furthermore, M41 reacted with a human recombinant Fab(Fab-d) that recognizes a conformational epitope on gp41 and binds HIV-1virions as well as HIV-1 infected cells but not uninfected cells asanalyzed by FACS. Deletion of either helix motif, i.e., DP107 or DP178,of the M41 fusion protein eliminated reactivity with Fab-d. Theseresults indicate that both helical regions, separated by 60 amino acidsin the primary sequence, are required to maintain the Fab-d epitope.

8.2.2. Anti-HIV Activity of the Recombinant Ectodomain of GP41

The wild type M41 fusion protein was tested for anti-HIV-1 activity. Asexplained, supra, synthetic peptides corresponding to the leucine zipper(DP107) and the C-terminal putative helix (DP178) show potent anti-HIVactivity. Despite inclusion of both these regions, the recombinant M41protein did not affect HIV-1 induced membrane fusion at concentrationsas high as 50 μM (Table XXV, below). TABLE XXV DISRUPTION OF THE LEUCINEZIPPER OF GP41 FREES THE ANTI-HIV MOTIF DP107 DP178 M41 M41-P M41-PΔ178Cell fusion 1 μM  1 nM >50 μM 83 nM >50 μM (IC₉₀) Fab-D — — 3.5 × 10⁻⁹2.5 × 10⁻⁸ — binding (k_(D)) HIV infectivity 1 μM 80 nM >16 μM 66 nM  >8μM (IC₉₀)1 The affinity constants of Fab-d binding to the fusion proteins weredetermined using a protocol described by B. Friguet et al., 1985, J.Immunol. Method. 77:305-319.— = No detectable binding of Fab-d to the fusion proteins.Antiviral Infectivity Assays. 20 μl of serially diluted virus stock wasincubated for 60 minutes at ambient temperature with 20 μl of theindicated concentration of purified recombinant fusion protein in RPMI1640 containing 10% fetal bovine serum and antibiotics in a 96-wellmicrotiter plate. 20 μl of CEM4 cells at 6 × 10⁵ cells/ml were added toeach well, and cultures were incubated at 37° C. in a humidified CO₂incubator. Cells were cultured for 9# days by the addition of fresh medium every 2 to 3 days. On days 5, 7,and 9 postinfection, supernatant samples were assayed for reversetranscriptase (RT) activity, as described below, to monitor viralreplication. The 50% tissue culture infectious dose (TCID₅₀) wascalculated for each condition according to the formula of Reed & Muench,1937, Am. J. Hyg. 27:493-497. RT activity was determined by amodification of the published methods of Goff et al., 1981, J. Virol.38:239-248 # as described in Chen et al., 1993, AIDS Res. HumanRetroviruses 9:1079-1086.

Surprisingly, a single amino acid substitution, proline in place ofisoleucine in the middle of the leucine zipper motif, yielded a fusionprotein (M41-P) which did exhibit antiviral activity (Table XXV and FIG.8). As seen in Table XXV, M41-P blocked syncytia formation by 90% atapproximately 85 nM and neutralized HIV-1_(IIIB) infection by 90% atapproximately 70 nM concentrations. The anti-HIV-1 activity of M41-Pappeared to be mediated by the C-terminal helical sequence sincedeletion of that region from M41-P yielded an inactive fusion protein,M41-PΔ178 (Table XXV). This interpretation was reinforced by experimentsdemonstrating that a truncated fusion protein lacking the DP178sequence, M41Δ178, abrogated the potent anti-fusion activity of theDP178 peptide in a concentration-dependent manner (FIG. 9). The sametruncated fusion protein containing the proline mutation disrupting theleucine zipper, M41-PΔ178, was not active in similar competitionexperiments (FIG. 9). The results indicate that the DP178 peptideassociates with a second site on gp41 whose interactive structure isdependent on a wild type leucine zipper sequence. A similar interactionmay occur within the wild type fusion protein, M41, and act to form anintramolecular clasp which sequesters the DP178 region, making itunavailable for anti-viral activity.

A specific association between these two domains is also indicated byother human monoclonal Fab-d studies. For example, Fab-d failed to bindeither the DP178 peptide or the fusion protein M41Δ178, but its epitopewas reconstituted by simply mixing these two reagents together (FIG.10). Again, the proline mutation in the leucine zipper domain of thefusion protein, M41-PΔ178, failed to reconstitute the epitope in similarmixing experiments.

9. EXAMPLE Method for Computer-Assisted Identification of DP107-Like andDP178-Like Sequences

A number of known coiled-coil sequences have been well described in theliterature and contain heptad repeat positioning for each amino acid.Coiled-coil nomenclature labels each of seven amino acids of a heptadrepeat A through G, with amino acids A and D tending to be hydrophobicpositions. Amino acids E and G tend to be charged. These four positions(A, D, E, and G) form the amphipathic backbone structure of a monomericalpha-helix. The backbones of two or more amphipathic helices interactwith each other to form di-, tri-, tetrameric, etc., coiled-coilstructures. In order to begin to design computer search motifs, a seriesof well characterized coiled coils were chosen including yeasttranscription factor GCN4, Influenza Virus hemagglutinin loop 36, andhuman proto-oncogenes c-Myc, c-Fos, and c-Jun. For each peptidesequence, a strict homology for the A and D positions, and a list of theamino acids which could be excluded for the B, C, E, F, and G positions(because they are not observed in these positions) was determined.Motifs were tailored to the DP107 and DP178 sequences by deducing themost likely possibilities for heptad positioning of the amino acids ofHIV-1 Bru DP-107, which is known to have coiled-coil structure, andHIV-1 Bru DP178, which is still structurally undefined. The analysis ofeach of the sequences is contained in FIG. 12. For example, the motiffor GCN4 was designed as follows:

-   1. The only amino acids (using standard single letter amino acid    codes) found in the A or D positions of GCN4 were [LMNV].-   2. All amino acids were found at B, C, E, F, and G positions except    {CFGIMPTW}.-   3. The PESEARCH motif would, therefore, be written as follows:

[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)-

[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)-

[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)-

[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)

Translating or reading the motif: “at the first A position either L, M,N, or V must occur; at positions B and C (the next two positions) accepteverything except C, F, G, I, M, P, T, or W; at the D position either L,M, N, or V must occur; at positions E, F, and G (the next 3 positions)accept everything except C, F, G, I, M, P, T, or W.” This statement iscontained four times in a 28-mer motif and five times in a 35-mer motif.The basic motif key then would be: [LMNV]-{CFGIMPTW}. The motif keys forthe remaining well described coiled-coil sequences are summarized inFIG. 12.

The motif design for DP107 and DP178 was slightly different than the28-mer model sequences described above due to the fact that heptadrepeat positions are not defined and the peptides are both longer than28 residues. FIG. 13 illustrates several possible sequence alignmentsfor both DP107 and DP178 and also includes motif designs based on28-mer, 35-mer, and full-length peptides. Notice that only slightdifferences occur in the motifs as the peptides are lengthened.Generally, lengthening the base peptide results in a less stringentmotif. This is very useful in broadening the possibilities foridentifying DP107- or DP-178-like primary amino acid sequences referredto in this document as “hits”.

In addition to making highly specific motifs for each type peptidesequence to be searched, it is also possible to make “hybrid” motifs.These motifs are made by “crossing” two or more very stringent motifs tomake a new search algorithm which will find not only both “parent” motifsequences but also any peptide sequences which have similarities to one,the other, or both “parents”. For example, in FIG. 14 the “parent”sequence of GCN4 is crossed with each of the possible “parent” motifs ofDP-107. Now the hybrid motif must contain all of the amino acids foundin the A and D positions of both parents, and exclude all of the aminoacids not found in either parent at the other positions. The resultinghybrid from crossing GCN4 or [LMNV]{CFGIMPTW} and DP107 (28-mer with thefirst L in the D position) or [ILQT]{CDFIMPST}, is [ILMNQTV]{CFIMPT}.Notice that now only two basic hybrid motifs exist which cover bothframing possibilities, as well as all peptide lengths of the parentDP-107 molecule. FIG. 15 represents the “hybridizations” of GCN4 withDP-178. FIG. 16 represents the “hybridizations” of DP107 and DP178. Itis important to keep in mind that the represented motifs, both parentand hybrid, are motif keys and not the depiction of the full-lengthmotif needed to actually do the computer search.

Hybridizations can be performed on any combination of two or moremotifs. FIG. 17 summarizes several three-motif hybridizations includingGCN4, DP107 (both frames), and DP178 (also both frames). Notice that theresulting motifs are now becoming much more similar to each other. Infact, the first and third hybrid motifs are actually subsets of thesecond and fourth hybrid motifs respectively. This means that the firstand third hybrid motifs are slightly more stringent than the second andfourth. It should also be noted that with only minor changes in thesefour motifs, or by hybridizing them, a single motif could be obtainedwhich would find all of the sequences. However, it should be rememberedthat stringency is also reduced. Finally, the most broad-spectrum andleast-stringent hybrid motif is described in FIG. 18 which summarizesthe hybridization of GCN4, DP107 (both frames), DP178 (both frames),c-Fos, c-Jun, c-Myc, and Flu loop 36.

A special set of motifs was designed based on the fact that DP-178 islocated only approximately ten amino acids upstream of the transmembranespanning region of gp41 and just C-terminal to a proline which separatesDP107 and DP178. It has been postulated that DP178 may be an amphipathichelix when membrane associated, and that the proline might aid in theinitiation of the helix formation. The same arrangement was observed inRespiratory Syncytial Virus; however, the DP178-like region in thisvirus also had a leucine zipper just C-terminal to the proline.Therefore, N-terminal proline-leucine zipper motifs were designed toanalyze whether any other viruses might contain this same pattern. Themotifs are summarized in FIG. 19.

The PC/Gene protein database contains 5879 viral amino acid sequences(library file PVIRUSES; CD-ROM release 11.0). Of these, 1092 are viralenveloped or glycoprotein sequences (library file PVIRUSE1). Tables Vthrough XIV contain lists of protein sequence names and motif hitlocations for all the motifs searched.

10. EXAMPLE Computer-Assisted Identification of DP107 and DP178-LikeSequences in Human Immunodeficiency Virus

FIG. 20 represents search results for HIV-1 BRU isolate gp41 (PC/Geneprotein sequence PENV_HV1BR). Notice that the hybrid motif which crossesDP-107 and DP-178 (named 107×178×4; the same motif as found in FIG. 16found three hits including amino acids 550-599, 636-688, and 796-823.These areas include DP-107 plus eight N-terminal and four C-terminalamino acids; DP178 plus seven N-terminal and ten C-terminal amino acids;and an area inside the transmembrane region (cytoplasmic). FIG. 20 alsocontains the results obtained from searching with the motif namedALLMOTI5, for which the key is found in FIG. 17 ({CDGHP} {CFP}×5). Thismotif also found three hits including DP107 (amino acids 510-599), DP178(615-717), and a cytoplasmic region (772-841). These hits overlap thehits found by the motif 107×178×4 with considerable additional sequenceson both the amino and carboxy termini. This is not surprising in that107×178×4 is a subset of the ALLMOTI5 hybrid motif. Importantly, eventhough the stringency of ALLMOTI5 is considerably less than 107×178×4,it still selectively identifies the DP107 and DP178 regions of gp41shown to contain sequences for inhibitory peptides of HIV-1. The resultsof these two motif searches are summarized in Table V under the PC/Geneprotein sequence name PENV HV1BR. The proline-leucine zipper motifs alsogave several hits in HIV-1 BRU including 503-525 which is at the veryC-terminus of gp12O, just upstream of the cleavage site (P7LZIPC andP12LZIPC); and 735-768 in the cytoplasmic domain of gp41 (P23LZIPC).These results are found in Tables VIII, IX, and X under the samesequence name as mentioned above. Notice that the only area of HIV-1 BRUwhich is predicted by the Lupas algorithm to contain a coiled-coilregion, is from amino acids 635-670. This begins eight amino acidsN-terminal to the start and ends eight amino acids N-terminal to the endof DP178. DP107, despite the fact that it is a known coiled coil, is notpredicted to contain a coiled-coil region using the Lupas method.

11. EXAMPLE Computer-Assisted Identification of DP107-like andDP178-like Sequences in Human Respiratory Syncytial Virus

FIG. 21 represents search results for Human Respiratory Syncytial Virus(RSV; Strain A2) fusion glycoprotein F1 (PC/Gene protein sequence namePVGLF_HRSVA). Motif 107×178×4 finds three hits including amino acids152-202, 213-243, and 488-515. The arrangement of these hits is similarto what is found in HIV-1 except that the motif finds two regions withsimilarities to DP-178, one just downstream of what would be called theDP107 region or amino acids 213-243, and one just upstream of thetransmembrane region (also similar to DP178) or amino acids 488-515.Motif ALLMOTI5 also finds three areas including amino acids 116-202,267-302, and 506-549. The proline-leucine zipper motifs also gaveseveral hits including amino acids 205-221 and 265-287 (PLLZIPC 265-280,P12LZIPC), and 484-513 (P7LZIPC and P12LZIPC 484-506, P23LZIPC). Noticethat the PLZIP motifs also identify regions which share locationsimilarities with DP-178 of HIV-1.

12. EXAMPLE Computer-Assisted Identification of DP107-Like andDP178-Like Sequences in Simian Immunodeficiency Virus

Motif hits for Simian immunodeficiency Virus gp41 (AGM3 isolate; PC/Geneprotein sequence name PENV_SIVAG) are shown in FIG. 22. Motif 107×178×4finds three hits including amino acids 566-593, 597-624, and 703-730.The first two hits only have three amino acids between them and couldprobably be combined into one hit from 566-624 which would represent aDP107-like hit. Amino acids 703 to 730 would then represent a DP178-likehit. ALLMOTI5 also finds three hits including amino acids 556-628(DP107-like), 651-699 (DP178-like), and 808-852 which represents thetransmembrane spanning region. SIV also has one region from 655-692 witha high propensity to form a coiled coil as predicted by the Lupasalgorithm. Both 107×178×4 and ALLMOTI5 motifs find the same region. SIVdoes not have any PLZIP motif hits in gp41.

The identification of DP178/DP107 analogs for a second SIV isolate(MM251) is demonstrated in the Example presented, below, in Section 19.

13. EXAMPLE Computer-Assisted Identification of DP107-Like and DP178Like Sequences in Canine Distemper Virus

Canine Distemper Virus (strain Onderstepoort) fusion glycoprotein F1(PC/Gene Protein sequence name PVGLF_CDVO) has regions similar to HumanRSV which are predicted to be DP107-like and DP178-like (FIG. 23). Motif107×178×4 highlights one area just C-terminal to the fusion peptide atamino acids 252-293. Amino acids 252-286 are also predicted to be coiledcoil using the Lupas algorithm. Almost 100 amino acids C-terminal to thefirst region is a DP178-like area at residues 340-367. ALLMOTI5highlights three areas of interest including: amino acids 228-297, whichcompletely overlaps both the Lupas prediction and the DP107-like107×178×4 hit; residues 340-381, which overlaps the second 107×178×4hit; and amino acids 568-602, which is DP178-like in that it is locatedjust N-terminal to the transmembrane region. It also overlaps anotherregion (residues 570-602) predicted by the Lupas method to have a highpropensity to form a coiled coil. Several PLZIP motifs successfullyidentified areas of interest including P6 and P12LZIPC which highlightresidues 336-357 and 336-361 respectively; P1 and P12LZIPC which findresidues 398-414; and P12 and P23LZIPC which find residues 562-589 and562-592 respectively.

14. EXAMPLE Computer-Assisted Identification of DP107-Like andDP178-Like Sequences in Newcastle Disease Virus

FIG. 24 shows the motif hits found in Newcastle Disease Virus (strainAustralia-Victoria/32; PC Gene protein sequence name PVGLF_NDVA). Motif107×178×4 finds two areas including a DP107-like hit at amino acids151-178 and a DP178-like hit at residues 426-512. ALLMOTI5 finds threeareas including residues 117-182, 231-272, and 426-512. The hits from426-512 include a region which is predicted by the Lupas method to havea high coiled-coil propensity (460-503). The PLZIP motifs identify onlyone region of interest at amino acids 273-289 (P1 and 12LZIPC).

15. EXAMPLE Computer-Assisted Identification of DP107-Like andDP178-Like Sequences in Human Parainfluenza Virus

Both motifs 107×178×4 and ALLMOTI5 exhibit DP107-like hits in the sameregion, 115-182 and 117-182 respectively, of Human Parainfluenza Virus(strain NIH 47885; PC/Gene protein sequence name PVGLF_p13H4; (FIG. 25).In addition, the two motifs have a DP178-like hit just slightlyC-terminal at amino acids 207-241. Both motifs also have DP178-like hitsnearer the transmembrane region including amino acids 457-497 and462-512 respectively. Several PLZIP motif hits are also observedincluding 283-303 (P5LZIPC), 283-310 (P12LZIPC), 453-474 (P6LZIPC), and453-481 (P23LZIPC). The Lupas algorithm predicts that amino acids122-176 may have a propensity to form a coiled-coil.

16. EXAMPLE Computer-Assisted Identification of DP107-Like andDP178-Like Sequences of Influenza a Virus

FIG. 26 illustrates the Lupas prediction for a coiled coil in InfluenzaA Virus (strain A/Aichi/2/68) at residues 379-436, as well as the motifhits for 107×178×4 at amino acids 387-453, and for ALLMOTI5 at residues380-456. Residues 383-471 (38-125 of HA2) were shown by Carr and Kim tobe an extended coiled coil when under acidic pH (Carr and Kim, 1993,Cell 73: 823-832). The Lupas algorithm predicts a coiled-coil atresidues 379-436. All three methods successfully predicted the regionshown to actually have coiled-coil structure; however, ALLMOTI5predicted the greatest portion of the 88 residue stretch.

17. EXAMPLE Potential Respiratory Syncytial Virus DP178/DP107 Analogs:CD and Antiviral Characterization

In the Example presented herein, respiratory syncytial virus (RSV)peptides identified by utilizing the computer-assisted search motifsdescribed in the Examples presented in Sections 9 and 11, above, weretested for anti-RSV activity. Additionally, circular dichroism (CD)structural analyses were conducted on the peptides, as discussed below.It is demonstrated that several of the identified peptides exhibitpotent antiviral capability. Additionally, it is shown that several ofthese peptides exhibit a substantial helical character.

17.1 Materials and Methods

Structural analyses: The CD spectra were measured in a 10 mM sodiumphosphate, 150 mM sodium chloride, pH 7.0, buffer at approximately 10 mMconcentrations, using a 1 cm pathlength cell on a Jobin/YvonAutodichrograph Mark V CD spectrophotometer. Peptides were synthesizedaccording to the methods described, above, in Section 6.1. Peptideconcentrations were determined from A₂₈₀ using Edlehoch's method (1967,Biochemistry 6:1948).

Anti-RSV antiviral activity assays: The assay utilized herein tested theability of the peptides to disrupt the ability of HEp2 cells acutelyinfected with RSV (i.e., cells which are infected with a multiplicity ofinfection of greater than 2) to fuse and cause syncytial formation on amonolayer of uninfected an uninfected line of Hep-2 cells. The lower theobserved level of fusion, the greater the antiviral activity of thepeptide was determined to be.

Uninfected confluent monolayers of Hep-2 cells were grown in microtiterwells in 3% EMEM (Eagle Minimum Essential Medium w/o L-glutamine [BioWhittaker Cat. No. 12-125F], with fetal bovine serum [FBS; which hadbeen heat inactivated for 30 minutes at 56° C.; Bio Whittaker Cat. No.14-501F) supplemented at 3%, antibiotics (penicillin/streptomycin; BioWhittaker Cat. No. 17-602E) added at 1%, and glutamine added at 1%.

To prepare Hep2 cells for addition to uninfected cells, cultures ofacutely infected Hep2 cells were washed with DPBS (Dulbecco's PhosphateBuffered Saline w/o calcium or magnesium; Bio Whittaker Cat. No.17-512F) and cell monolayers were removed with Versene (1:5000; GibcoLife Technologies Cat. No. 15040-017). The cells were spun 10 minutesand resuspended in 3% FBS. Cell counts were performed using ahemacytometer. Persistent cells were added to the uninfected Hep-2cells.

The antiviral assay was conducted by, first, removing all media from thewells containing uninfected Hep-2 cells, then adding peptides (at thedilutions described below) in 3% EMEM, and 100 acutely RSV-infected Hep2cells per well. Wells were then incubated at 37° C. for 48 hours.

After incubation, cells in control wells were checked for fusioncenters, media was removed from the wells, followed by addition, to eachwell, of either Crystal Violet stain or XTT. With respect to CrystalViolet, approximately 50 μl 0.25% Crystal Violet stain in methanol wereadded to each well. The wells were rinsed immediately, to remove excessstain, and were allowed to dry. The number of syncytia per well werethen counted, using a dissecting microscope.

With respect to XTT(2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilideinner salt), 50 μl XTT (1 mg/ml in RPMI buffered with 100 mM HEPES, pH7.2-7.4, plus 5% DMSO) were added to each well. The OD_(450/690) wasmeasured (after blanking against growth medium without cells orreagents, and against reagents) according to standard procedures.

Peptides: The peptides characterized in the study presented herein were:

1) peptides T-142 to T-155 and T-575, as shown in FIG. 27A, and peptidesT-22 to T-27, T-68, T-334 and T-371 to T-375 and T-575, as shown in FIG.27B;

2) peptides T-120 to T-141 and T-576, as shown in FIG. 27B, and peptidesT-12, T-13, T-15, T-19, T-28 to T-30, T-66, T-69, T-70 and T-576, asshown in FIG. 27D; and

3) peptides T-67 and T-104 to T-119 and T-384, as shown in FIG. 28A, andpeptides T-71, T-613 to T-617, T-662 to T-676 and T-730, as shown inFIG. 28B.

The peptides of group 1 represent portions of the RSV F2 proteinDP178/107-like region. The peptides of group 2 represent portions of theRSV F1 protein DP107-like region. The peptides of groups 3 representportions of the RSV F1 protein DP178-like region.

Each peptide was tested at 2-fold serial dilutions ranging from 100μg/ml to approximately 100 ng/ml. For each of the assays, a wellcontaining no peptide was also used. The IC₅₀ data for each peptiderepresents the average of several experiments conducted utilizing thatpeptide.

17.2 Results

The data summarized in FIGS. 27A-B and 28A-B represent antiviral andstructural information obtained from peptides derived from the RSV F2DP178/DP107-like F2 region (FIG. 27A-B), the RSV F1 DP-107-like region(FIG. 27C-D) and the RSV DP178-like F2 region (FIG. 28A-B).

As shown in FIGS. 27A-D, a number of the RSV DP178/DP107-like peptidesexhibited a detectable level of antiviral activity. Peptides from theRSV DP178/DP107-like F2 region (FIG. 27A-B), for example, T-142 to T-145and T-334 purfied peptides, exhibited detectable levels of antiviralactivity, as evidenced by their IC₅₀ values. Further, a number of RSV F1DP107-like peptides (FIG. 27C-D) exhibited a sizable level of antiviralactivity as purified peptides, including, for example, peptides T-124 toT-127, T-131, T-135 and T-137 to T-139, as demonstrated by their lowIC₅₀ values. In addition, CD analysis FIGS. 27A, 27C) reveals that manyof the peptides exhibit some detectable level of helical structure.

The results summarized in FIG. 28A-B demonstrate that a number ofDP178-like purified peptides exhibit a range of potent anti-viralactivity. These peptides include, for example, T-67, T-104, T-105 andT-107 to T-119, as listed in FIG. 28A, and T-665 to T-669 and T-671 toT-673, as listed in FIG. 28B. In addition, some of the DP178-likepeptides exhibited some level of helicity.

Thus, the computer assisted searches described, hereinabove,successfully identified viral peptide domains that represent highlypromising anti-RSV antiviral compounds.

18. EXAMPLE Potential Human Parainfluenza Virus Type 3 DP178/DP107Analogs: CD and Antiviral Characterization

In the Example presented herein, human parainfluenza virus type 3(HPIV3) peptides identified by utilizing the computer-assisted searchmotifs described in the Examples presented in Sections 9 and 15, above,were tested for anti-HPIV3 activity. Additionally, circular dichroism(CD) structural analyses were conducted on the peptides, as discussedbelow. It is demonstrated that several of the identified peptidesexhibit potent antiviral capability. Additionally, it is shown thatseveral of these peptides exhibit a substantial helical character.

18.1 Materials and Methods

Structural analyses: Structural analyses consisted of circular dichroism(CD) studies. The CD spectra were measured in a 10 mM sodium phosphate,150 mM sodium chloride, pH 7.0, buffer at approximately 10 mMconcentrations, using a 1 cm pathlength cell on a Jobin/YvonAutodichrograph Mark V CD spectrophotometer. Peptide concentrations weredetermined from A₂₈₀ using Edlehoch's method (1967, Biochemistry6:1948).

Anti-HPIV3 antiviral activity assays: The assay utilized herein testedthe ability of the peptides to disrupt the ability of Hep2 cellschronically infected with HPIV3 to fuse and cause syncytial formation ona monolayer of an uninfected line of CV-1W cells. The more potent thelower the observed level of fusion, the greater the antiviral activityof the peptide.

Uninfected confluent monolayers of CV-1W cells were grown in microtiterwells in 3% EMEM (Eagle Minimum Essential Medium w/o L-glutamine [BioWhittaker Cat. No. 12-125F], with fetal bovine serum [FBS; which hadbeen heat inactivated for 30 minutes at 56° C.; Bio Whittaker Cat. No.14-501F) supplemented at 3%, antibiotics/antimycotics (Gibco BRL LifeTechnologies Cat. No. 15040-017) added at 1%, and glutamine added at 1%.

To prepare Hep2 cells for addition to uninfected cells, cultures ofchronically infected Hep2 cells were washed with DPBS (Dulbecco'sPhosphate Buffered Saline w/o calcium or magnesium; Bio Whittaker Cat.No. 17-512F) and cell monolayers were removed with Versene (1:5000;Gibco Life Technologies Cat. No. 15040-017). The cells were spun 10minutes and resuspended in 3% FBS. Cell counts were performed using ahemacytometer. Persistent cells were added to the uninfected CV-1Wcells.

The antiviral assay was conducted by, first, removing all media from thewells containing uninfected CV-1W cells, then adding peptides (at thedilutions described below) in 3% EMEM, and 500 chronicallyHPIV3-infected Hep2 cells per well. Wells were then incubated at 37° C.for 24 hours.

On day 2, after cells in control wells were checked for fusion centers,media was removed from the wells, followed by addition, to each well, ofapproximately 50 μl 0.25% Crystal Violet stain in methanol. Wells wererinsed immediately, to remove excess stain and were then allowed to dry.The number of syncytia per well were then counted, using a dissectingmicroscope.

Alternatively, instead of Crystal Violet analysis, cells were assayedwith XTT, as described, avove, in Section 17.1.

Peptides: The peptides characterized in the study presented herein were:

-   1) Peptides 157 to 188, as shown in FIG. 29A, and peptides T-38 to    T-40, T-42 to T-46 and T-582, as shown in FIG. 29B. These peptides    are derived from the DP107 region of the HPIV3 F1 fusion protein    (represented by HPF3 107, as shown in FIG. 29A); and-   2) Peptides 189 to 210, as shown in FIG. 30A, and T-269, T-626,    T-383 and T-577 to T-579, as shown in FIG. 30B. These peptides are    primarily derived from the DP178 region of the HPIV3 F1 fusion    protein (represented by HPF3 178, as shown in FIG. 30A). Peptide    T-626 contains two mutated amino acid resides (represented by a    shaded background). Additionally, peptide T-577 represents F1 amino    acids 65-100, T-578 represents F1 amino acids 207-242 and T-579    represents F1 amino acids 273-309.

Each peptide was tested at 2-fold serial dilutions ranging from 500 g/mlto approximately 500 ng/ml. For each of the assays, a well containing nopeptide was also used.

18.2 Results

The data summarized in FIGS. 29A-C and 30A-B represent antiviral andstructural information obtained from peptides derived from the HPIV3fusion protein DP107-like region (FIG. 29A-C) and the HPIV3 fusionprotein DP178-like region (FIG. 30A-B).

As shown in FIG. 29A-B, a number of the HPIV3 DP107-like peptidesexhibited potent levels of antiviral activity. These peptides include,for example, peptides T-40, T-172 to T-175, T-178, T-184 and T-185.

CD analysis reveals that a number of the peptides exhibit detectable tosubstantial level of helical structure. The CD spectra for one of thepeptides, 184, which exhibits substantial helicity is summarized in FIG.29C.

The results summarized in FIG. 30A-B demonstrate that a number of theDP178-like peptides tested exhibit a range of anti-viral activity. Thesepeptides include, for example, peptides 194 to 211, as evidenced bytheir low IC₅₀ values. In fact, peptides 201 to 205 exhibit IC₅₀ valuesin the nanogram/ml range. In addition, many of the DP178-like peptidesexhibited some level of helicity.

Thus, the computer assisted searches described, hereinabove, havesuccessfully identified viral peptide domains that represent highlypromising anti-HPIV3 antiviral compounds.

19. EXAMPLE Computer-Assisted Identification of DP178/DP107 Analogs inSimian Immunodeficiency Virus

FIG. 31 represents search results for SIV isolate MM251 (PC/Genesprotein sequence PENV_SIVM2). Both 107×178×4 and ALLMOTI5 search motifsidentified two regions with similarities to DP107 and/or DP178.

The peptide regions found by 107×178×4 were located at amino acidresidues 156-215 and 277-289. The peptide regions found by ALLMOTI5 werelocated at amino acid residues 156-219 and 245-286. Both motifs,therefore, identify similar regions.

Interestingly, the first SIV peptide region (i.e., from amino acidresidue 156 to approximately amino acid residue 219) correlates with aDP107 region, while the second region identified (i.e., fromapproximately amino acid residue 245 to approximately amino acid residue289) correlates with the DP178 region of HIV. In fact, an alignment ofSIV isolate MM251 and HIV isolate BRU, followed by a selection of thebest peptide matches for HIV DP107 and DP178, reveals that the bestmatches are found within the peptide regions identified by the 107×178×4and ALLMOTI5 search motifs.

It should be noted that a potential coiled-coil region at amino acidresidues 242-282 is predicted by the Lupas program. This is similar tothe observation in HIV in which the coiled-coil is predicted by theLupas program to be in the DP178 rather than in the DP107 region. It ispossible, therefore, that SIV may be similar to HIV in that it maycontain a coiled-coil structure in the DP107 region, despite such astructure being missed by the Lupas algorithm. Likewise, it may be thatthe region corresponding to a DP178 analog in SIV may exhibit anundefined structure, despite the Lupas program's prediction of acoiled-coil structure.

20. EXAMPLE Computer-Assisted Identification of DP178/DP107 Analogs inEpstein-Barr Virus

The results presented herein describe the identification of DP178/DP107analogs within two different Epstein-Barr Virus proteins. Epstein-Barris a human herpes virus which is the causative agent of, for example,infectious mononucleosis (IM), and is also associated withnasopharyngeal carcinomas (NPC), Burkitt's lymphoma and other diseases.The virus predominantly exists in the latent form and is activated by avariety of stimuli.

FIG. 32 depicts the search motif results for the Epstein-Barr Virus(Strain B95-8; PC/Gene® protein sequence PVGLB_EBV) glycoprotein gp110precursor (gp115). The 107×178×4 motif identified two regions ofinterest, namely the regions covered by amino acid residues 95-122 and631-658. One PZIP region was identified at amino acid residue 732-752which is most likely a cytoplasmic region of the protein. The Lupasalgorithm predicts a coiled-coil structure for amino acids 657-684. NoALLMOTI5 regions were identified.

FIG. 33 depicts the search motif results for the Zebra (or EB1)trans-activator protein (BZLF1) of the above-identified Epstein-Barrvirus. This protein is a transcription factor which represents theprimary mediator of viral reactivation. It is a member of the b-ZIPfamily of transcription factors and shares significant homology with thebasic DNA-binding and dimerization domains of the cellular oncogenesc-fos and C/EBP. The Zebra protein functions as a homodimer.

Search results demonstrate that the Zebra protein exhibits a singleregion which is predicted to be either of DP107 or DP178 similarity, andis found between the known DNA binding and dimerization regions of theprotein. Specifically, this region is located at amino acid residues193-220, as shown in FIG. 33. The Lupas program predicted no coiled-coilregions.

21. EXAMPLE Computer-Assisted Identification of DP178/DP107 Analogs inMeasles Virus

FIG. 34 illustrates the motif search results for the fusion protein F1of measles virus, strain Edmonston (PC Genes protein sequencePVGLF_MEASE), successfully identifying DP178/DP107 analogs.

The 107×178×4 motif identifies a single region at amino acid residues228-262. The ALLMOTI5 search motif identifies three regions, includingamino acid residues 116-184, 228-269 and 452-500. Three regionscontaining proline residues followed by a leucine zipper-like sequencewere found beginning at proline residues 214, 286 and 451.

The Lupas program identified two regions it predicted had potential forcoiled-coil structure, which include amino acid residues 141-172 and444-483.

22. EXAMPLE Computer-Assisted Identification of DP178/DP107 Analogs inHepatitis B Virus

FIG. 35 depicts the results of a PZIP motif search conducted on theHepatitis B virus subtype AYW. Two regions of interest within the majorsurface antigen precursor S protein were identified. The first lies justC-terminal to the proposed fusion peptide of the major surface antigen(Hbs) which is found at amino acid residues 174-191. The second regionis located at amino acid residues 233-267. The Lupas program predicts nocoiled-coil repeat regions.

In order to test the potential anti-HBV antiviral activity of theseD178/DP107 analog regions, peptides derived from area around the analogregions are synthesized, as shown in FIG. 52A-B. These peptidesrepresent one amino acid peptide “walks” through the putativeDP178/DP107 analog regions. The peptides are synthesized according tostandard Fmoc chemistry on Rinkamide MBHA resins to provide for carboxyterminal blockade (Chang, C. D. and Meinhofer, J., 1978, Int. J. Pept.Protein Res. 11:246-249; Fields, G. B. and Noble, R. L., 1990, Int. J.Pept. Protein Res. 35:161-214). Follwing complete synthesis, the peptideamino-terminus is blocked through automated acetylation and the peptideis cleaved with trifluoroacetic acid (TFA) and the appropriatescavengers (King, D. S. et al., 1990, Int. J. Pept. Res. 36:255-266).After cleavage, the peptide is precipitated with ether and dried undervacuum for 24 hours.

The anti-HBV activity of the peptides is tested by utilizing standardassays to determine the test peptide concentration required to cause anacceptable (e.g., 90%) decrease in the amount of viral progeny formed bycells exposed to an HBV viral inoculum. Candidate antivial peptides arefurther characterized in model systems such as wood chuck tissue cultureand animal sytems, prior to testing on humans.

23. EXAMPLE Computer-Assisted Identification of DP178/DP107 Analogs inSimian Mason-Pfizer Monkey Virus

The results depicted herein illustrate the results of search motifsconducted on the Simian Mason-Pfizer monkey virus. The motifs revealDP178/DP107 analogs within the enveloped (TM) protein GP20, as shown inFIG. 36.

The 107×178×4 motifs identifies a region at amino acid residues 422-470.The ALLMOTI5 finds a region at amino acid residues 408-474. The Lupasprogram predicted a coiled-coil structure a amino acids 424-459.

24. EXAMPLE Computer-Assisted Identification of DP178/DP107 Analogs inBacterial Proteins

The results presented herein demonstrate the identification ofDP178/DP107 analogs corresponding to sequences present in proteins of avariety of bacterial species.

FIG. 37 depicts the search motif results for the Pseudomonas aeruginosafimbrial protein (Pilin). Two regions were identified by motifs107×178×4 and ALLMOTI5. The regions located at amino acid residues 30-67and 80-144 were identified by the 107×178×4 motif. The regions at aminoacid residues 30-68 and 80-125 were identified by the ALLMOTI5.

FIG. 38 depicts the search motif results for the Pseudomonas gonorrhoeaefimbrial protein (Pilin). A single region was identified by both the107×178×4 and the ALLMOTI5 motifs. The region located at amino acidresidues 66-97 was identified by the 107×178×4 motif. The region locatedat amino acid residues 66-125 were identified by the ALLMOTI5 searchmotif. No coiled-coil regions were predicted by the Lupas program.

FIG. 39 depicts the search motif results for the Hemophilus Influenzafimbrial protein (Pilin). A single region was identified by both the107×178×4 and the ALLMOTI5 motifs. The region located at amino acidresidues 102-129 was identified by the 107×178×4 motif. The regionlocated at amino acid residues 102-148 were identified by the ALLMOTI5search motif. No coiled-coil regions were predicted by the Lupasprogram.

FIG. 40 depicts the search motif results for the Staphylococcus aureustoxic shock syndrome Hemophilus Influenza fimbrial protein (Pilin). Asingle region was identified by both the 107×178×4 and the ALLMOTI5motifs. The region located at amino acid residues 102-129 was identifiedby the 107×178×4 motif. The region located at amino acid residues102-148 were identified by the ALLMOTI5 search motif. No coiled-coilregions were predicted by the Lupas program.

FIG. 41 summarizes the motif search results conducted on theStaphylococcus aureus enterotoxin Type E protein. These resultsdemonstrate the successful identification of DP178/DP107 analogscorresponding to peptide sequences within this protein, as describedbelow.

The ALLMOTI5 motif identified a region at amino acid residues 22-27. The107×178×4 motif identified two regions, with the first at amino acidresidues 26-69 and the second at 88-115. A P12LZIPC motif searchidentified two regions, at amino acid residues 163-181 and 230-250.

The Lupas program predicted a region with a high propensity for coilingat amino acid residues 25-54. This sequence is completely containedwithin the first region identified by both ALLMOTI5 and 107×178×4motifs.

FIG. 42 depicts the search motif results conducted on a secondStaphylococcus aureus toxin, enterotoxin A. Two regions were identifiedby the ALLMOTI5 motif, at amino acid residues 22-70 and amino acidresidues 164-205. The 107×178×4 motif found two regions, the first atamino acid residues 26-69 and the second at amino acid residues 165-192.A P23LZIPC motif search revealed a region at amino acid residues216-250. No coiled-coil regions were predicted by the Lupas program.

FIG. 43 shows the motif search results conducted on the E. coli heatlabile enterotoxin A protein, demonstrating that identification ofDP178/DP107 analogs corresponding to peptides located within thisprotein. Two regions were identified by the ALLMOTI5 motif, with thefirst residing at amino acid residues 55-115, and the second residing atamino acid residues 216-254. The 107×178×4 motif identified a singleregion at amino acid residues 78-105. No coiled-coil regions werepredicted by the Lupas program.

25. EXAMPLE Computer-Assisted Identification of DP178/DP107 Analogswithin Various Human Proteins

The results presented herein demonstrate the identification ofDP178/DP107 analogs corresponding to peptide sequences present withinseveral different human proteins.

FIG. 44 illustrates the search motif results conducted on the humanc-fos oncoprotein. The ALLMOTI5 motif identified a single region atamino acid residues 155-193. The 107×178×4 motif identified one regionat amino acid residues 162-193. The Lupas program predicted a region atamino acid residues 148-201 to have coiled-coil structure.

FIG. 45 illustrates the search motif results conducted on the humanlupus KU autoantigen protein P70. The ALLMOTI5 motif identified a singleregion at amino acid residues 229-280. The 107×178×4 motif identifiedone region at amino acid residues 235-292. The Lupas program predicted aregion at amino acid residues 232-267 to have coiled-coil structure.

FIG. 46 illustrates the search motif results conducted on the human zincfinger protein 10. The ALLMOTI5 motif identified a single region atamino acid residues 29-81. The 107×178×4 motif identified one region atamino acid residues 29-56. A P23LZIPC motif search found a single regionat amino acid residues 420-457. The Lupas program predicted nocoiled-coil regions.

26. EXAMPLE Potential Measles Virus DP178/DP107 Analogs: CD andAntiviral Characterization

In the Example presented herein, measles (MeV) virus DP178-like peptidesidentified by utilizing the computer-assisted search motifs described inthe Examples presented in Sections 9 and 21, above, are tested foranti-MeV activity. Additionally, circular dichroism (CD) structuralanalyses are conducted on the peptides, as discussed below. It isdemonstrated that several of the identified peptides exhibit potentantiviral capability. Additionally, it is shown that none of the thesepeptides exhibit a substantial helical character.

26.1 Materials and Methods

Structural analyses: The CD spectra were measured in a 10 mM sodiumphosphate, 150 mM sodium chloride, pH 7.0, buffer at approximately 10 mMconcentrations, using a 1 cm pathlength cell on a Jobin/YvonAutodichrograph Mark V CD spectrophotometer. Peptide concentrations weredetermined from A₂₈₀ using Edlehoch's method (1967, Biochemistry6:1948).

Anti-MeV antiviral activity syncytial reduction assay: The assayutilized herein tested the ability of the peptides to disrupt theability of Vero cells acutely infected with MeV (i.e., cells which areinfected with a multiplicity of infection of 2-3) to fuse and causesyncytial formation on a monolayer of an uninfected line of Vero cells.The more potent the peptide, the lower the observed level of fusion, thegreater the antiviral activity of the peptide.

Uninfected confluent monolayers of Vero cells were grown in microtiterwells in 10% FBS EMEM (Eagle Minimum Essential Medium w/o L-glutamine[Bio Whittaker Cat. No. 12-125F], with fetal bovine serum [FBS; whichhad been heat inactivated for 30 minutes at 56° C.; Bio Whittaker Cat.No. 14-501F) supplemented at 10%, antibiotics/antimycotics (BioWhittaker Cat. No. 17-602E) added at 1%, and glutamine added at 1%.

To prepare acutely infected Vero cells for addition to the uninfectedcells, cultures of acutely infected Vero cells were washed twice withHBSS (Bio Whittaker Cat. No. 10-543F) and cell monolayers were removedwith trypsin (Bio Whittaker Cat. No. 17-161E). Once cells detached,media was added, any remaining clumps of cells were dispersed, andhemacytometer cell counts were performed.

The antiviral assay was conducted by, first, removing all media from thewells containing uninfected Vero cells, then adding peptides (at thedilutions described below) in 10% FBS EMEM, and 50-100 acutelyMeV-infected Vero cells per well. Wells were then incubated at 37° C.for a maximum of 18 hours.

On day 2, after cells in control wells were checked for fusion centers,media was removed from the wells, followed by addition, to each well, ofapproximately 50 μl 0.25% Crystal Violet stain in methanol. Wells wererinsed twice with water immediately, to remove excess stain and werethen allowed to dry. The number of syncytia per well were then counted,using a dissecting microscope.

Anti-MeV antiviral activity plaque reduction assay: The assay utilizedherein tested the ability of the peptides to disrupt the ability of MeVto infect permissive, uninfected Vero cells, leading to the infectedcells' fusing with uninfected cells to produce syncytia. The lower theobserved level of syncytial formation, the greater the antiviralactivity of the peptide.

Monolayers of uninfected Vero cells are grown as described above.

The antiviral assay was conducted by, first, removing all media from thewells containing uninfected Vero cells, then adding peptides (at thedilutions described below) in 10% FBS EMEM, and MeV stock virus at afinal concentration of 30 plaque forming units (PFU) per well. Wellswere then incubated at 37° C. for a minimum of 36 hours and a maximum of48 hours.

On day 2, after cells in control wells were checked for fusion centers,media was removed from the wells, followed by addition, to each well, ofapproximately 50 μl 0.25% Crystal Violet stain in methanol. Wells wererinsed twice with water immediately, to remove excess stain and werethen allowed to dry. The number of syncytia per well were then counted,using a dissecting microscope.

Peptides: The peptides characterized in the study presented herein werepeptides T-252A0 to T-256A0, T-257B1/C1, and T-258B1 to T-265B0, andT-266A0 to T-268A0, as shown in FIG. 47. These peptides represent a walkthrough the DP178-like region of the MeV fusion protein.

Each peptide was tested at 2-fold serial dilutions ranging from 100μg/ml to approximately 100 ng/ml. For each of the assays, a wellcontaining no peptide was also used.

26.2 Results

The data summarized in FIG. 47 represents antiviral and structuralinformation obtained via “peptide walks” through the DP178-like regionof the MeV fusion protein.

As shown in FIG. 47, the MeV DP178-like peptides exhibited a range ofantiviral activity as crude peptides. Several of these peptides werechosen for purification and further antiviral characterization. The IC₅₀values for such peptides were determined, as shown in FIG. 47, andranged from 1.35 μg/ml (T-257B1/C1) to 0.072 μg/ml (T-265B1). None ofthe DP178-like peptides showed, by CD analysis, a detectable level ofhelicity.

Thus, the computer assisted searches described, hereinabove, as in forexample, the Example presented in Section 9, for example, successfullyidentified viral peptide domains that represent highly promisinganti-MeV antiviral compounds.

27. EXAMPLE POTENTIAL SIV DP178/DP107 Analogs Antiviral Characterization

In the Example presented herein, simian immunodeficiency virus (SIV)DP178-like peptides identified by utilizing the computer-assisted searchmotifs described in the Examples presented in Sections 9, 12 and 19,above, were tested for anti-SIV activity. It is demonstrated thatseveral of the identified peptides exhibit potent antiviral capability.

27.1 Materials and Methods

Anti-SIV antiviral assays: The assay utilized herein were as reported inLangolis et al. (Langolis, A. J. et al., 1991, AIDS Research and HumanRetroviruses 7:713-720).

Peptides: The peptides characterized in the study presented herein werepeptides T-391 to T-400, as shown in FIG. 48. These peptides represent awalk through the DP178-like region of the SIV TM protein.

Each peptide was tested at 2-fold serial dilutions ranging from 100 g/mlto approximately 100 ng/ml. For each of the assays, a well containing nopeptide was also used.

27.2 Results

The data summarized in FIG. 48 represents antiviral information obtainedvia “peptide walks” through the DP178-like region of the SIV TM protein.

As shown in FIG. 48, peptides T-391 to T-400 were tested and exhibited apotent antiviral activity as crude peptides.

Thus, the computer assisted searches described, hereinabove, as in forexample, the Example presented in Section 9, for example, successfullyidentified viral peptide domains that represent highly promisinganti-SIV antiviral compounds.

28. EXAMPLE Anti-Viral Activity of DP107 and DP-178 Peptide Truncationsand Mutations

The Example presented in this Section represents a study of theantiviral activity of DP107 and DP178 truncations and mutations. It isdemonstrated that several of these DP107 and DP178 modified peptidesexhibit substantial antiviral activity.

28.1 Materials and Methods

Anti-HIV assays: The antiviral assays performed were as those described,above, in Section 6.1. Assays utilized HIV-1/IIIb and/or HIV-2 NIHZisolates. Purified peptides were used, unless otherwise noted in FIGS.49A-C.

Peptides: The peptides characterized in the study presented herein were:

-   -   1) FIGS. 49A-C present peptides derived from the region around        and containing the DP178 region of the HIV-1 BRU isolate.        Specifically, this region spanned from gp41 amino acid residue        615 to amino acid residue 717. The peptides listed contain        truncations of this region and/or mutations which vary from the        DP178 sequence amino acid sequence. Further, certain of the        peptides have had amino- and/or carboxy-terminal groups either        added or removed, as indicated in the figures; and    -   2) FIG. 50. presents peptides which represent truncations of        DP107 and/or the gp41 region surrounding the DP107 amino acid        sequence of HIV-1 BRU isolate. Certain of the peptides are        unblocked or biotinylated, as indicated in the figure.

Blocked peptides contained an acyl N-terminus and an amido C-terminus.

28.2 Results

Anti-HIV antiviral data was obtained with the group 1 DP178-derivedpeptides listed in FIG. 49A-C. The full-length, non-mutant DP178 peptide(referred to in FIG. 49A-C as T20) results shown are for 4 ng/ml.

In FIG. 49A, a number of the DP178 truncations exhibited a high level ofantiviral activity, as evidenced by their low IC₅₀ values. Theseinclude, for example, test peptides T-50, T-624, T-636 to T-641, T-645to T-650, T-652 to T-654 and T-656. T-50 represents a test peptide whichcontains a point mutation, as indicated by the residue's shadedbackground. The HIV-1-derived test peptides exhibited a distinctstrain-specific antiviral activity, in that none of the peptides testedon the HIV-2 NIHZ isolate demonstrated appreciable anti-HIV-2 antiviralactivity.

Among the peptides listed in FIG. 49B, are test peptides representingthe amino (T-4) and carboxy (T-3) terminal halves of DP178 were tested.The amino terminal peptide was not active (IC₅₀>400 μg/ml) whereas thecarboxy terminal peptide showed potent antiviral activity (IC₅₀=3μg/ml). A number of additional test peptides also exhibited a high levelof antiviral activity. These included, for example, T-61/T-102, T-217 toT-221, T-235, T-381, T-677, T-377, T-590, T-378, T-591, T-271 to T-272,T-611, T-222 to T-223 and T-60/T-224. Certain of the antiviral peptidescontain point mutations and/or amino acid residue additions which varyfrom the DP178 amino acid sequence.

In FIG. 49C, point mutations and/or amino and/or carboxy-terminalmodifications are introduced into the DP178 amino acid sequence itself.As shown in the figure, the majority of the test peptides listed exhibitpotent antiviral activity.

Truncations of the DP107 peptide (referred to in IG. 50 as T21) werealso produced and tested, as shown in FIG. 50. FIG. 50 also presentsdata concerning blocked and unblocked peptides which contain additionalamino acid residues from the gp41 region in which the DP107 sequenceresides. Most of these peptides showed antiviral activity, as evidencedby their low IC₅₀ values.

Thus, the results presented in this Section demonstrate that not only dothe full length DP107 and DP178 peptides exhibit potent antiviralactivity, but truncations and/or mutant versions of these peptides canalso possess substantial antiviral character.

29: EXAMPLE Potential Epstein-Barr DP178/DP107 Analogs: AntiviralCharacterization

In the Example presented herein, peptides derived from the Epstein-Barr(EBV) DP-178/DP107 analog region of the Zebra protein identified, above,in the Example presented in Section 20 are described and tested foranti-EBV activity. It is demonstrated that among these peptides are oneswhich exhibit potential anti-viral activity.

29.1 Materials and Methods

Electrophoretic Mobility Shift Assays (EMSA): Briefly, an EBV Zebraprotein was synthesized utilizing SP6 RNA polymerase in vitrotranscription and wheat germ in vitro translation systems (PromegaCorporation recommendations; Butler, E. T. and Chamberlain, M. J., 1984,J. Biol. Chem. 257:5772; Pelham, H. R. B. and Jackson, R. J., 1976, Eur.J. Biochem. 67:247). The in vitro translated Zebra protein was thenpreincubated with increasing amounts of peptide up to 250 ng/ml prior tothe addition of 10,000 to 20,000 c.p.m. of a ³²P-labeled Zebra responseelement DNA fragment. After a 20 minute incubation in the presence ofthe response element, the reaction was analyzed on a 4% non-denaturingpolyacrylamide gel, followed by autoradiography, utilizing standardgel-shift procedures. The ability of a test peptide to prevent Zebrahomodimer DNA binding was assayed by the peptide's ability to abolishthe response element gel migration retardation characteristic of aprotein-bound nucleic acid molecule.

Peptides: The peptides characterized in this study represent peptidewalks through the region containing, and flanked on both sides by, theDP178/DP107 analog region identified in the Example presented in Section20, above, and shown as shown in FIG. 33. Specifically, the peptidewalks covered the region from amino acid residue 173 to amino acidresidue 246 of the EBV Zebra protein.

Each of the tested peptides were analyzed at a range of concentrations,with 150 ng/ml being the lowest concentration at which any of thepeptides exerted an inhibitory effect.

29.2 Results

The EBV Zebra protein transcription factor contains a DP178/DP107 analogregion, as demonstrated in the Example presented, above, in Section 20.This protein appears to be the primary factor responsible for thereactivation capability of the virus. A method by which the DNA-bindingfunction of the Zebra virus may be abolished may, therefore, representan effective antiviral technique. In order to identify potentialanti-EBV DP178/DP107 peptides, therefore, peptides derived from theregion identified in Section 20, above, were tested for their ability toinhibit Zebra protein DNA binding.

The test peptides' ability to inhibit Zebra protein DNA binding wasassayed via the EMSA assays described, above, in Section 28.1. The datasummarized in FIG. 51A-B presents the results of EMSA assays of thelisted EBV test peptides. These peptides represent one amino acid“walks” through the region containing, and flanked on both sides by, theDP178/DP107 analog region identified in the Example presented in Section20, above, and shown as shown in FIG. 33. As shown in FIG. 51A-B, theregion from which these peptides are derived lies from EBV Zebra proteinamino acid residue 173 to 246. A number of the test peptides which wereassayed exhibited an ability to inhibit Zebra protein homodimer DNAbinding, including 439, 441, 444 and 445.

Those peptides which exhibit an ability to inhibit Zebra protein DNAbinding represent potential anti-EBV antiviral compounds whose abilityto inhibit EBV infection can be further characterized.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims. LENGTHY TABLEThe patent application contains a lengthy table section. A copy of thetable is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070037141A1)An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An isolated peptide recognized by an ALLMOTI5, 107×178×4 or a PLZIPsequence search motif.
 2. The peptide of claim 1 wherein the peptidecorresponds to a peptide present in a virus.
 3. The peptide of claim 2in which the virus is HIV-1 or HIV-2.
 4. The peptide of claim 2 in whichthe virus is a respiratory syncytial virus.
 5. The peptide of claim 2 inwhich the virus is a human parainfluenza virus.
 6. The peptide of claim2 in which the virus is an influenza virus.
 7. The peptide of claim 2 inwhich the virus is a hepatitis B virus.
 8. The peptide of claim 2wherein the virus is an Epstein-Barr virus.
 9. A method for theinhibition of transmission of a virus to a cell, comprising contactingthe cell with an effective concentration of a peptide recognized by anALLMOTI5, 107×178×4 or a PLZIP sequence search motif for an effectiveperiod of time so that no infection of the cell by the virus occurs. 10.The method of claim 9 wherein the virus is HIV-1 or HIV-2.
 11. Themethod of claim 9 wherein the virus is a respiratory syncytial virus.12. The method of claim 9 wherein the virus is a human parainfluenzavirus.
 13. The method of claim 9 wherein the virus is an influenzavirus.
 14. The method of claim 9 in which the virus is a hepatitis Bvirus.
 15. The method of claim 9 wherein the virus is an Epstein-Barrvirus.